Glucose Utilization In Cells Research Articles

Cellular mechanisms of insulin resistance, lipodystrophy and atherosclerosis induced by HIV protease inhibitors

Accumulating clinical evidence now links HIV protease inhibitors (HPIs) to the pathogenesis of insulin resistance, dyslipidaemia, lipodystrophy and atherosclerosis associated with highly active anti-retroviral therapy. Here we briefly describe the evidence for a distinct causative role for HPIs, and explore the cellular mechanisms proposed to underlie these side-effects. Acute inhibition of GLUT4-mediated glucose transport, and defective insulin signalling induced by chronic exposure to nelfinavir, are described as cellular mechanisms of insulin resistance. Interference with adipogenesis and adipocyte apoptosis and nelfinavir-induced activation of lipolysis are discussed as potential mechanisms of HPI-induced lipodystrophy. HPI-induced free radical production, apoptosis and increased glucose utilization in vascular smooth muscle cells are presented as possible novel mechanisms for atherosclerosis. Common pathways and cause-effect relationships between the various cellular mechanisms presented are then discussed, with emphasis on the role of insulin resistance, free radical production and enhanced lipolysis. Understanding the cellular mechanisms of HPI-induced side-effects will enhance the search for improved anti-retroviral therapy, and may also shed light on the pathogenesis of common forms of insulin resistance, dyslipidaemia and atherosclerosis.

A Hydroxychalcone Derived from Cinnamon Functions as a Mimetic for Insulin in 3T3-L1 Adipocytes

Objectives: These studies investigated the ability of a hydroxychalcone from cinnamon to function as an insulin mimetic in 3T3-L1 adipocytes.Methods: Comparative experiments were performed with the cinnamon methylhydroxychalcone polymer and insulin with regard to glucose uptake, glycogen synthesis, phosphatidylinositol-3-kinase dependency, glycogen synthase activation and glycogen synthase kinase-3β activity. The phosphorylation state of the insulin receptor was also investigated.Results: MHCP treatment stimulated glucose uptake and glycogen synthesis to a similar level as insulin. Glycogen synthesis was inhibited by both wortmannin and LY294002, inhibitors directed against the PI-3-kinase. In addition, MHCP treatment activated glycogen synthase and inhibited glycogen synthase kinase-3β activities, known effects of insulin treatment. Analysis of the insulin receptor demonstrated that the receptor was phosphorylated upon exposure to the MHCP. This supports that the insulin cascade was triggered by MHCP. Along with comparing MHCP to insulin, experiments were done with MHCP and insulin combined. The responses observed using the dual treatment were greater than additive, indicating synergism between the two compounds.Conclusion: Together, these results demonstrate that the MHCP is an effective mimetic of insulin. MHCP may be useful in the treatment of insulin resistance and in the study of the pathways leading to glucose utilization in cells.

Physiological concentrations of insulin augment pancreatic cancer cell proliferation and glucose utilization by activating MAP kinase, PI3 kinase and enhancing GLUT-1 expression.

Pancreatic carcinoma is characterized by poor prognosis and lack of response to conventional therapy for reasons that are not clear. Because of the structural relationship between the exocrine and endocrine pancreas and high concentrations of islet hormones bathing pancreatic tissue, we hypothesized that pancreatic cancer cell proliferation and glucose utilization are regulated by pancreatic islet hormones, particularly insulin. Based on this, the effect of islet hormones on pancreatic cancer cells in vitro was investigated. Five pancreatic cancer cell lines, CD11, CD18, HPAF, PANC-1, and MiaPaCa2 were used to investigate the effect of islet hormones on cell proliferation, glucose utilization, and GLUT-1 expression. Insulin, but not somatostatin and glucagon, induced pancreatic cancer cell growth in a concentration- and time-dependent manner. At concentrations within the range of those in the intrapancreatic vasculature, insulin (10(-10)-10(-8) mol/L) markedly increased [3H]-thymidine incorporation. Insulin significantly enhanced glucose utilization of pancreatic cancer cells before it enhanced cell proliferation. The MAPK kinase inhibitor PD 098059 abolished insulin-stimulated DNA synthesis and partially reduced insulin-stimulated glucose uptake. In contrast, the PI3 kinase inhibitor wortmannin substantially inhibited insulin-induced glucose uptake and partially blocked thymidine incorporation. Furthermore, after 24-hour treatment with insulin, GLUT-I expression in pancreatic cancer cells was markedly increased, indicating that insulin enhances glucose utilization partly through increasing glucose transport. These findings suggest that insulin stimulates proliferation and glucose utilization in pancreatic cancer cells by two distinct pathways. Insulin augments DNA synthesis mainly by MAP kinase activation and glucose uptake mainly by PI3 kinase activation and enhancement of GLUT-I expression. High intrapancreatic concentrations of insulin are likely to play an important role in stimulating pancreatic cancer growth indirectly by increasing substrate availability as well as by direct action as a trophic factor.

Effects of lipoic acid on glucose utilization, protein glycosylation and viscosity of high glucose-treated red blood cells

UTILIZATION, PROTEIN GLYCOSYLATION AND VISCOSITY OF HIGH GLUCOSE-TREATED RED BLOOD CELLS Sushi1 K. Jain, Melissa Palmer, Yanyun Chen Department of Pediatrics, Louisiana State University Medical Center, Shreveport, LA 71130 USA Glycosylation of proteins can cause inactivation of enzymes and alterations in the structure and function of collagen and membranes. which may play a role in the long-term complications of diabetes and aging. Lipoic acid has been shown beneficial in reducing diabetic complications. such as. neuropathy. The present study was undertaken to test the hypothesis that lipoic acid increases glucose utilization and thereby reduces protein glycosylation and viscosity of red blood cells (RBC). RBC (15% hematocrit in PBS) were treated with high glucose levels (6-45 mM) with and without lipoic acid (5-200 LM) for 24 hrs in a shakine water bath at 3?‘C. Glucose was ‘determined using gl&ose oxidasel Glycosylated hemoglobin (HbAI) was determined using affinity chromatography to assess protein glycosylation. an: viscositv of washed RBC at tixed hematocrit was determined using computerized Brookfield viscometer. Lipoic acid treatment increased glucose utilization in high glucose (45 mM) exposed RBC by 75-120%. Over untreated fresh RBC, the increase in HbA, level in high glucose treated RBC was reduced by 22-40% in the presence of lipoic acid compared with high glucose alone. Treatment of RBC with high glucose increased viscosity of RBC. However. presence of lipoic acid in the medium significantly blocked increase in viscosity of high glucose-treated RBC. The level of glycated hemoglobin in blood has been considered to reflect the degree of glycation of other proteins that are freely exposed to circulating glucose. Using REX as a model. this study demonstrates that lipoic acid can reduce glycosylation of proteins and viscosity of cells exposed to high glucose. LIPID PEROXIDATION AND ANTIOXIDANT ENZYMES IN IMMUNE COMPLEX DISEASE G. Wbjcicka, A. Marciniak, D. G6my, R. CzabakGarbacz’, A. Korolczuk’, J. Beltowski Dept of Pathophysiologv, ‘Dept of Physiology, 2Dept of Pathomorphology, University of Medicine, Lublin, Poland The purpose of this study was to investigate lipid peroxidation and antioxidant mechanisms during early stage of immunisation and developed type III hypersensitivity. The male New Zeland rabbits (2.5 3 kg) were immunised by i.v. BSA. Four days after BSA administration malonyldialdehyde (MDA) level increased in plasma, renal cortex and renal medulla by 63%, 246% and 140% respectively, conjugated diens (CDs) increased in plasma, renal cortex and renal medulla by 76%, 122% and 63% respectively, lipid hydroperoxides (HPE) increased in plasma by 14%, in renal cortex by 11% and in renal medulla by 85%. Superoxide dismutase (SOD) activity in erythrocytes increased by 17%, blood glutathione peroxidase (Gpx) increased by 63% and total antioxidant status (TAS) decreased by 19%. Proteinuria was maximal 8 days after BSA administration. This day we observed 110% increase in MDA level in renal cortex and 271% increase in renal medulla. CDs increased by 60% in plasma, by 23% in renal cortex and by 40% in renal medulla. HPE in plasma was increased by 20% and in renal cortex by 100%. SOD and Gpx increased by 61% and 57% respectively whereas TAS decreased by 45%.

Hypoxia and CYP1A1 induction-dependent regulation of proteins involved in glucose utilization in Caco-2 cells.

Although induction of cytochrome P-450 1A1 (CYP1A1) in the Caco-2 clone TC7 alters glucose utilization and modifies the expression of sucrase-isomaltase (SI) and hexose transporters, nothing is known of the events that control these effects. In this study, we analyzed the effects of beta-naphthoflavone (beta-NF) and hypoxia on these parameters and expression of key enzymes of glucose metabolism. Both beta-NF and hypoxia induce similar changes: 1) induction of CYP1A1 mRNA; 2) increased glucose consumption and lactic acid production and lower glycogen content; 3) downregulation of SI and upregulation of GLUT1 mRNAs; 4) downregulation of fructose-1,6-bisphosphatase and pyruvate kinase mRNAs and upregulation of phosphoenolpyruvate carboxykinase, pyruvate dehydrogenase, lactate dehydrogenase, and phosphofructokinase mRNAs; and 5) upregulation of c-fos and c-jun mRNAs. Although addition of inhibitors of CYP1A1 catalytic activity to beta-NF-treated cells totally inhibits the enzyme activity, it does not modify CYP1A1 mRNA response and associated effects, thus excluding a direct role for the enzyme per se. These results point to a possible physiological implication of the signal-transduction pathway responsible for CYP1A1 induction.

Regulation of energy metabolism by neurotransmitters in astrocytes in primary culture and in an immortalized cell line.

Evidence suggests that astrocytes might play an important role in cerebral energy metabolism. A recently developed cell line, called DI TNC1, displays several characteristic features of astrocytes. Thus, we have investigated in these cells a number of parameters related to energy metabolism. First, glycogen, the major energy reserve in the brain, is present in these cells and its levels are influenced by the glucose content of the growth medium and the presence of serum. Second, several neurotransmitters including noradrenaline and vasoactive intestinal peptide (VIP) induce a glycogenolytic response. Their effect on glycogen is paralleled by a similar effect on the formation of cyclic AMP, which is presumably the second messenger involved. Third, noradrenaline stimulates glucose utilization (as reflected by 2-deoxyglucose uptake) in DI TNC1 cells, an effect which is mimicked by the second messenger arachidonate. Interestingly, two actions of neurotransmitters, which are well characterized in primary astrocytes, are absent in DI TNC1 cells. These are the noradrenaline- and VIP-induced resynthesis of glycogen and the glutamate-stimulated glycolysis. In summary, the observations reported here lend further support to the concept that astrocytes are important for the control of brain energy metabolism. In addition, DI TNC1 cells might represent an interesting preparation to help decipher some of the astrocytic functions related to energy metabolism.

Effects of aging on the activities of pyruvate dehydrogenase complex and its kinase in rat heart

Effects of aging on the activities of heart pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase were examined using 7, 35 and 60 wk old rats. Aging did not affect the total activity of pyruvate dehydrogenase complex but decreased the activity state (percentage of active form) of the complex in rats under the fed condition (52%, 36% and 26% for 7, 35 and 60 wk old rats, respectively). This decrease in the complex activity with aging was suggested to be associated with an age-related decrease in the blood glucose disposal. Starvation for 24 h decreased the activity state to less than 3% in all of the age groups. The activity of pyruvate dehydrogcnase kinase associated with the complex was not related to the alteration in the activity state of the complex; the kinase activity was slightly lower in 60 wk old rats than in the younger rats under the fed condition and activation of the kinase by starvation was greater in the younger rats. The mechanism for the decrease in activity of pyruvate dehydrogenase complex was discussed on the basis of glucose and fatty acid utilization of heart muscle cells.

The glucose sensor protein glucokinase is expressed in glucagon-producing alpha-cells.

Expression of glucokinase in hepatocytes and pancreatic 6-cells is of major physiologic importance to mammalian glucose homeostasis. Liver glucokinase catalyzes the first committed step in the disposal of glucose, and beta-cell glucokinase catalyzes a rate-limiting step required for glucose-regulated insulin release. The present study reports the expression of glucokinase in rat glucagon-producing alpha-cells, which are negatively regulated by glucose. Purified rat alpha-cells express glucokinase mRNA and protein with the same transcript length, nucleotide sequence, and immunoreactivity as the beta-cell isoform. Glucokinase activity accounts for more than 50% of glucose phosphorylation in extracts of alpha-cells and for more than 90% of glucose utilization in intact cells. The glucagon-producing tumor MSL-G-AN also contained glucokinase mRNA, protein, and enzymatic activity. These data indicate that glucokinase may serve as a metabolic glucose sensor in pancreatic alpha-cells and, hence, mediate a mechanism for direct regulation of glucagon release by extracellular glucose. Since these cells do not express Glut2, we suggest that glucose sensing does not necessarily require the coexpression of Glut2 and glucokinase.

Neurotrophins and Alzheimer's disease: Beyond the cholinergic neurons

Improvement of the cholinergic deficit in Alzheimer's disease (AD) by intracerebral application of nerve growth factor (NGF) serves as a paradigmatic example for a novel approach to the treatment of neurodegeneration. The first part of this paper presents and discusses experiments which were performed in our laboratory to study the NGF receptor response after intracerebral NGF treatment in vivo. We found that intraparenchymal NGF elicits prolonged tyrosine phosphorylation of Trk type NGF receptors. Our results indicate that intraparenchymal injections are preferable to intraventricular application for targeting specific neuronal populations with minimal side effects. Besides the cholinergic deficit, severely disturbed brain energy metabolism, particularly in cortical association areas, is another consistent feature of AD. Metabolic hypofunction is observed early in the disease progression and correlates with the cognitive impairment. Cell culture findings are presented which indicate that brain-derived neurotrophic factor (BDNF), and other neurotrophins with activity on the TrkB tyrosine kinase receptor, increase mRNA levels and biochemical activity of enzymes of the glycolytic pathway in brain cells. Treatment with these factors was also found to stimulate glucose utilization in rat embryonic cortex cells in primary cultures. Our observations suggest that selected neurotrophins should become useful not only for the treatment of the cholinergic deficit in AD, but also of the cortical metabolic hypofunction associated with this disease.

Citrinin affects the oxidative metabolism of BHK-21 cells.

The effects of citrinin on energy production along the respiratory chain and on glycolytic lactate production were examined in BHK-21 cultured cells. Citrinin inhibited the oxygen consumption rate by about 45 per cent. The respiratory rate of digitonin-treated cells energized with succinate, in the presence of ADP, was reduced by about 39 per cent. The mycotoxin inhibited the glucose utilization of BHK-21 cells by about 86 per cent. Cells treated with citrinin produced a small quantity of pyruvate, but were unable to produce lactate. It is concluded that BHK-21 cells cannot generate lactate when oxidative metabolism is inhibited by citrinin. The perturbations in BHK-21 cells caused by citrinin are due to alterations in mitochondrial function and in the glycolytic anaerobic pathway.

Regulation of Hexokinase II Gene Transcription and Glucose Phosphorylation by Catecholamines, Cyclic AMP, and Insulin

The hexokinases, by converting glucose to glucose-6-phosphate, help maintain the downhill gradient that results in movement of glucose into cells through the facilitative glucose transporters. GLUT4 and hexokinase (HK) II are the major transporter and hexokinase isoforms in skeletal muscle, heart, and adipose tissue, wherein insulin promotes glucose utilization. To understand whether hormones influence the contribution of phosphorylation to cellular glucose utilization, we investigated the effects that catecholamines, cyclic AMP (cAMP), and insulin have on HKII gene expression in cells representative of muscle (L6 cells) and brown (BFC-1B cells) and white (3T3-F442A cells) adipose tissues. Isoproterenol or the cAMP analog 8-chlorophenylthio-cAMP selectively increase HKII gene transcription in L6 cells, as does insulin (Printz RL, Koch S, Potter LP, O'Doherty RM, Tiesinga JJ, Moritz S, Granner DK: Hexokinase II mRNA and gene structure, regulation by insulin, and evolution. J Biol Chem 268:5209-5219, 1993), and cause a concentration- and time-dependent increase of HKII mRNA in both muscle and fat cell lines without changing HKI mRNA. Isoproterenol and insulin also increase the rate of synthesis of HKII protein and increase glucose phosphorylation and glucose utilization in L6 cells.

Metabolic and energetic changes during apoptosis in neural cells.

Changes in cellular energetic and metabolic parameters were analyzed at several time points during apoptosis of differentiated PC12 cells following removal of nerve growth factor (NGF). As approximately 60% of the population died during the period of study (24 h), most of the measured metabolic indicators declined over time. However, this decline paralleled the overall decrease in cellular viability, suggesting that, in individual cells, a compromised metabolic state occurred suddenly and very late in the death process. For example, when expressed as a function of viable cells, protein and RNA synthesis did not decrease until 24 h. Glucose utilization in live cells was never significantly reduced relative to control levels; lactate production decreased slightly within 4-8 h after NGF removal, but eventually rebounded to 122% of control levels by 24 h. ATP levels dropped 27% in an early predeath period, but then returned to near control levels (on a per-live-cell basis) once the population actively began to die. The ATP/ADP ratio remained at least 84% of control throughout. UTP/UDP and GTP/GDP ratios did not change significantly at any time point.

Coupled Glucose Transport and Metabolism in Cultured Neuronal Cells: Determination of the Rate-Limiting Step

In brain and nerves the phosphorylation of glucose, rather than its transport, is generally considered the major rate-limiting step in metabolism. Since little is known regarding the kinetic coupling between these processes in neuronal tissues, we investigated the transport and phosphorylation of [2-3H]glucose in two neuronal cell models: a stable neuroblastoma cell line (NCB20), and a primary culture of isolated rat dorsal root ganglia cells. When transport and phosphorylation were measured in series, phosphorylation was the limiting step, because intracellular glucose concentrations were the same as those outside of cells, and because the apparent Km for glucose utilization was lower than expected for the transport step. However, the apparent Km was still severalfold higher than the Km of hexokinase I. When [2-3H]glucose efflux and phosphorylation were measured from the same intracellular glucose pool in a parallel assay, rates of glucose efflux were three- to-fivefold greater than rates of phosphorylation. With the parallel assay, we observed that activation of glucose utilization by the sodium channel blocker veratridine caused a selective increase in glucose phosphorylation and was without effect on glucose transport. In contrast to results with glucose, both cell types accumulated 2-deoxy-D-[14C]glucose to concentrations severalfold greater than extracellular concentrations. We conclude from these studies that glucose utilization in neuronal cells is phosphorylation-limited, and that the coupling between transport and phosphorylation depends on the type of hexose used.

Glucokinase expression in rat hepatoma cells induces glucose uptake and is rate limiting in glucose utilization

In contrast to hepatocytes, hepatoma cells lack glucokinase activity and show increased aerobic glycolysis. FTO-2B and H4IIE rat hepatoma cell lines were obtained in which the rat glucokinase gene was expressed (FTOGK and H4GK). These lines were generated by infection of the hepatoma cells with a retroviral vector carrying the phosphoenolpyruvate carboxykinase (PEPCK)-glucokinase chimeric gene. Both the FTOGK and H4GK cells expressed the chimeric gene in a regulated manner, like the endogenous PEPCK gene. Glucokinase activity was detected in both FTOGK and H4GK. These cells lines showed a marked increase in glucose uptake with 18.5 mM glucose in the incubation medium. FTOGK and H4GK showed an increase in the content of glucose 6-phosphate, and were able to accumulate high levels of glycogen, in contrast to FTO-2B cells, which were unable to store the polysaccharide. In addition, cells expressing glucokinase showed high concentration of fructose 2,6-bisphosphate and substantial lactate production, which was related to the glucose concentration in the medium and the time of incubation. These results suggest that glucose phosphorylation is rate limiting for glucose uptake and utilization in FTO-2B and H4IIE cells.

Inhibition of potentially lethal damage recovery by altered pH, glucose utilization and proliferation in plateau growth phase human glioma cells.

Recovery from potentially lethal damage (PLD) has been measured in plateau growth phase human glioblastoma cells (U-87MG) under four postirradiation medium conditions. Recovery was maximal in depleted medium at an acidic pH, conditions which inhibit cellular proliferation. Compared with this control, PLD recovery (PLDR) was increasingly inhibited by alkalization of the existing medium (to pH 7.4), exchanging the old medium with fresh medium-pH acidified (to pH 6.8), and exchanging the old medium with fresh medium-pH unaltered (pH 7.4), respectively. These three medium adjustments were made at the time of irradiation. Increased glucose utilization (glycolysis) was detected postirradiation in all three cases, while increased proliferation was detected only when fresh medium was exchanged for old medium. Thus inhibition of PLDR has been correlated with increased glycolysis and increased proliferation during the recovery period. When acting together, these two processes provided almost complete inhibition. This study was revealed that the degree of inhibition may be related to the amount of glycolysis and/or proliferation occurring during the recovery period. Examining, in vitro, the range of PLDR achieved by postirradiation manipulation of medium pH may provide some indication of the range in PLDR that may be expected in vivo. Our study demonstrates that the effect of pH on glycolysis and proliferation may be important when determining the ability of a particular cell type to recover from PLD.

In vivo metabolic response of hepatic nonparenchymal cells and leukocytes to granulocyte-macrophage colony-stimulating factor.

This study investigates the in vivo glucose utilization of various immune-competent cells after an intra-arterial injection of a nonlethal dose (30 micrograms/kg body weight) of murine recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF). Injection of GM-CSF resulted in a rapid but transient reduction in the number of circulating neutrophils. After 20 min the number of neutrophils returned to normal values, and by 4 h it was about 80% greater than in time-matched saline-injected controls. One hour after the treatment, neutrophils were accumulated in the livers of GM-CSF-injected animals but not in control livers. In vivo glucose utilization by circulating neutrophils and mononuclear cells and various liver cell types was investigated by combining the 2-deoxyglucose tracer technique with cell isolation procedures. GM-CSF increased the in vivo glucose utilization of circulating and infiltrating neutrophils by more than 200%. Glucose utilization by circulating mononuclear cells was also doubled. After GM-CSF injection, glucose utilization by Kupffer cells was increased by 130% and by hepatic endothelial cells was increased by 60%. Indomethacin pretreatment blunted the hyperglycemia caused by GM-CSF injection; however, it did not inhibit the increased glucose utilization by immune-competent cells. This suggests that the effect of GM-CSF on glucose utilization by these cells is not mediated by prostanoids and is at least partially independent of the mass action of elevated glucose concentration. These findings indicate that GM-CSF may be an important member of the cytokine cascade that mediates the acute in vivo metabolic response of immune-competent cells in sepsis or endotoxemia.

Histochemical evaluation of energy metabolism in rat glioma.

The key enzymes of oxidative phosphorylation and glycolysis were evaluated histochemically in rat-implanted C6 gliomas using spot densitometry. Hexokinase, the initial enzyme for the glycolysis pathway, was 40% higher within tumour than the contralateral cerebral cortex. A similar increase within tumours for 2-deoxyglucose was observed by autoradiography. Glucose-6-phosphate dehydrogenase (G6PDH), which is the first enzyme in the pentose phosphate pathway, shunting glucose towards nucleic acid synthesis, was more than 300% higher in gliomas compared with the normal cortex. In contrast, enzymes in the energy producing tricarboxylic acid cycle (succinate-, isocitrate-, and malate-dehydrogenase) and in the electron-transport system (cytochrome c oxidase) were significantly reduced in tumour (58% less than the contralateral cortex). Lactate dehydrogenase activity, which converts pyruvate to lactate, was 50% higher within tumour. Significant reductions of enzymatic activities also occurred in non-neoplastic tissue in ipsilateral hemisphere, with larger tumours. Some enzymes showed heterogeneous activity within tumours, especially G6PDH. These results suggest that: (1) energy production is more dependent on lactate production than on oxidative phosphorylation in C6 glioma, and (2) a significant part of the increased glucose utilization in glioma cells is due to increased activity of the pentose phosphate shunt for increased DNA synthesis, and not energy production.

Lactate production is the major metabolic fate of glucose in splenocytes and is altered in spontaneously diabetic BB rats

Enhanced glucose metabolism is necessary to support the activation and proliferation of lymphocytes. To define further quantitatively the metabolic fates of glucose and assess glucose utilization both in normal cells and in an autoimmune disease with abnormal lymphocytes, [U-14C]glucose conversion into 14CO2 and the production of lactate and pyruvate were measured in splenocytes. Cells from non-diabetes-prone (BBn) and spontaneously diabetic (BBd) rats were studied both freshly isolated 'resting' and cultured for 96 h with and without concanavalin A (Con A) stimulation. (1) Lactate was confirmed to be the major end product in both freshly isolated (53% of utilized glucose) and unstimulated cultured (62% of utilized glucose) cells from BBn animals studied at (2-8) x 10(6) cells/ml concentration. The use of concentrations from 10 x 10(6) to 300 x 10(6) cells/ml resulted in progressively less lactate production per 10(6) splenocytes. (2) Cells from BBd animals after stimulation with Con A incorporated less [3H]thymidine and produced significantly less lactate (155 +/- 14 versus 305 +/- 24 nmol/2 h per 10(6) cells) than did BBn cells (P less than 0.05). (3) However, more lactate (101 +/- 8 versus 78 +/- 6 nmol/5 h per 10(6) cells) was produced by 'resting' cells from BBd animals compared with BBn (P less than 0.03), and this difference was sustained after 4 days in culture. (4) Significantly greater amounts of pyruvate were produced by BBd than by BBn cells, particularly when stimulated with Con A, suggesting an alteration in the availability of reducing equivalents in BBd cells. (5) These results are consistent with prior metabolic as well as immunological 'activation' of cells in vivo in the BB diabetic animals.

Anomeric preference of glucose utilization in human erythrocytes loaded with glucokinase

Human erythrocytes were loaded with homogeneous rat liver glucokinase by an encapsulation method based on hypotonic hemolysis and isotonic resealing. As assayed at 10 mM glucose, glucokinase and hexokinase activities in glucokinase-loaded erythrocytes were 218 and 384 nmol/min/gHb, respectively; whereas hexokinase activity in both intact and unloaded red cells, which contain no glucokinase activity, was about 400 nmol/min/gHb. No difference in the rate of lactate production from glucose anomers between intact and unloaded erythrocytes suggested that the encapsulation procedure itself did not affect glucose utilization in red cells. Alpha-anomeric preference in lactate production from glucose was observed in glucokinase-loaded erythrocytes, whereas the beta anomer of glucose was more rapidly utilized than the alpha anomer in intact and unloaded erythrocytes. The results indicate that the step of glucose phosphorylation determines the anomeric preference in glucose utilization by human erythrocytes, since glucokinase and hexokinase are alpha- and beta-preferential, respectively, in glucose phosphorylation.

Galanin and Epinephrine Act on Distinct Receptors to Inhibit Insulin Release by the Same Mechanisms Including an Increase in K+Permeability of the B-Cell Membrane*

The mechanisms by which galanin and epinephrine affect pancreatic B-cell function were studied in normal mouse islets. In the presence of 15 mM glucose and 2.5 mM Ca2+, galanin (50 nM) and epinephrine (100 nM) hyperpolarized the B-cell membrane and suppressed electrical activity only transiently. These changes were accompanied by a decrease in 86Rb+ efflux from islet cells and nearly complete inhibition of insulin release. Both agents also decreased 86Rb+ efflux in the absence of Ca2+. Low concentrations (10-15 microM) of diazoxide, an activator of ATP-sensitive K+ channels, mimicked some effects of galanin and epinephrine. However, insulin release was more markedly inhibited by galanin or epinephrine than by diazoxide when electrical activity was similarly decreased, and diazoxide had no effect on 86Rb+ efflux in the absence of Ca2+. When the permeability to K+ was increased by 100 microM diazoxide and the hyperpolarization reversed by high extracellular K+, galanin and epinephrine still inhibited insulin release, but did not affect the membrane potential or 86Rb+ efflux. Galanin and epinephrine decreased glucose utilization and oxidation in islet cells by about 10%, whereas diazoxide had no effect. Blockade of alpha 2-adrenoceptors by yohimbine suppressed the effects of epinephrine, but not those of galanin. It is concluded that activation of galanin and alpha2-adrenergic receptors inhibits insulin release by the same mechanisms. These may involve an increase in K+ permeability of the B-cell membrane by opening ATP-sensitive K+ channels and an additional effect independent of the membrane potential.