Apolipoprotein J (clusterin) activates rodent microglia in vivo and in vitro.

Apolipoprotein J (apoJ; also known as clusterin and sulfated glycoprotein (SGP)-2) is associated with senile plaques in degenerating regions of Alzheimer's disease brains, where activated microglia are also prominent. We show a functional link between apoJ and activated microglia by demonstrating that exogenous apoJ activates rodent microglia in vivo and in vitro. Intracerebroventricular infusion of purified human plasma apoJ ( approximately 4 microg over 28 days) activated parenchymal microglia to a phenotype characterized by enlarged cell bodies and processes (phosphotyrosine immunostaining). In vitro, primary rat microglia were also activated by apoJ, with changes in morphology and induction of major histocompatibility complex class II (MHCII) antigen. ApoJ increased the secretion of reactive nitrogen intermediates in a dose-dependent manner (EC(50) 112 nm), which was completely blocked by aminoguanidine (AG), a nitric oxide synthase inhibitor. However, AG did not block the increased secretion of tumor necrosis factor-alpha by apoJ (EC(50) 55 nm). Microglial activation by apoJ was also blocked by an anti-apoJ monoclonal antibody (G7), and by chemical cleavage of apoJ with 2-nitro-5-thiocyanobenzoate. The mitogen-activated protein kinase kinase and protein kinase C inhibitors PD98059 and H7 inhibited apoJ-mediated induction of reactive nitrogen intermediate secretion from cultured microglia. As a functional measure, apoJ-activated microglia secreted neurotoxic agents in a microglia-neuron co-culture model. We hypothesize that ApoJ contributes to chronic inflammation and neurotoxicity through direct effects on microglia.

Glucose-glucose 6-phosphate cycling in hepatocytes determined by incorporation of 3HOH and D2O. Effect of glycosyns and fructose.

The phosphorylation of glucose and recycling between glucose and glucose-6-P was determined in hepatocytes from fasted rats by a novel method. The cells were incubated with [U-14C]glucose as sole substrate in media containing 3HOH and D2O. Recycling was calculated from the yield of protons in glucose and glycogen. Results with 3HOH and D2O were identical. Phosphorylation was obtained as the sum of recycling plus the U-14C yields in products. At 10 mM glucose, more than 4 out of 5 molecules of glucose-6-P were recycled. About 1.2 mumol of glucose min/g of liver was phosphorylated, 1 mumol was recycled, and 0.2 mumol was glycolyzed. The effect of two phenacylimidazolium compounds (designated as glycosyns) and low concentrations of fructose (0.05-0.2 mM) on phosphorylation and recycling were examined. The glycosyns doubled glucose uptake, mainly as glycogen, nearly abolished glycolysis, and decreased recycling from 80 to 50-60%. There was little change in phosphorylation. Fructose doubled the yield of tritium from [2-3H]glucose in short term incubations (20-30 min), confirming the results of Van Schaftingen ((1993) Diabetologia 36, 582-588). The effect was transient, and cells became refractory to fructose. There was no glycogen synthesis and little effect on recycling. A new phenacylimidazolium compound stimulated glycogen synthesis and suppressed glycolysis and recycling, like the compound designated as proglycosyn by Yamanuchi et al. ((1992) Arch. Biochem. Biophys. 294, 609-615). This new compound (glycosyn-2) was fully active at lower concentrations (maximal effect at 0.02 mM).

A concentration gradient of glucose from liver to plasma.

Concentrations of glucose in plasma water and liver water were determined in rats under a number of conditions. In fasted and fed postadsorptive rats, the concentration of glucose in plasma water averaged 5.5 +/- 0.5 and 6.8 +/- 0.2 mmol/L, respectively. The concentration in liver water was 8.2 +/- 0.9 and 11.1 +/- 1.1 mmol/L, respectively (mean +/- SD). The concentration ratio of liver water to plasma water (LW/PW) was 1.52 +/- 0.1 (range, 1.4 to 1.7; n = 7) in fasted rats and 1.64 +/- 0.39 (range, 1.38 to 2.0; n = 10 [mean +/- SD]) in fed rats. Fasted rats treated with glucagon contained no liver glycogen. The concentration of glucose in plasma water was 7.1 +/- 0.3 mmol/L and in liver water 9.2 +/- 0.5 mmol/L, and the LW/PW averaged 1.35 +/- 0.08 (range, 1.23 to 1.45; n = 8). Insulin-injected hypoglycemic rats contained very little glycogen. The LW/PW was 2.7 +/- 0.2. In fasted rats infused intragastrically with 30 mg/min/kg glucose, the concentration of glucose in mixed portal-arterial plasma water entering the liver averaged 10.3 +/- 0.8 mmol/L and that in liver water 12.4 +/- 1.3 mmol/L (mean +/- SD), with the LW/PW averaging 1.21 +/- 0.1 (range, 1.08 to 1.4; n = 8). Fasted rats were infused with 3.4 mg/min/kg sodium lactate labeled with 14C and 13C and with a trace amount of [3-3H]glucose. The glucose concentration in arterial plasma water averaged 6.4 +/- 1.0 mmol/L and in liver water 14.6 +/- 4.4 mmol/L (mean +/- SD).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of D2O on glycolysis by rat hepatocytes.

1. The effect of incorporating D2O into the incubation medium on glycolysis and gluconeogenesis by hepatocytes from fasted rats was examined. 2. The substitution by heavy water, D2O, at concentrations from 10 to 40%, stimulated glucose uptake, lactate production and CO2 yields from glucose. At 10 mM glucose, 40% D2O doubled glucose uptake, increased CO2 production by 40%, and increased lactate production by 350%. 3. The stimulation of lactate production decreased at higher glucose concentrations, but was still substantial even at 80 mM glucose. 4. There was no effect on CO2 production above glucose concentrations of 30 mM. 5. Ten percent D2O showed little inhibition of lactate uptake, its oxidation and gluconeogenesis. At 40% D2O the inhibition ranged from 10 to 20%. 6. No effect of D2O on the rate of glucokinase or glucose-6-phosphatase was observed. 7. The concentration of fructose, 2,6-P was not affected by D2O.

Stimulation of glycogen synthesis by proglycosyn (LY177507) by isolated hepatocytes of normal and streptozotocin diabetic rats.

The phenacylimidazolium compound LY177507 was shown by Harris et al. (Harris, R. A., Yamanuchi, K., Roach, P. J., Yen, T. T., Dominiani, S. J., and Stephens, T. W. (1989) J. Biol. Chem. 264, 14674-14680) to stimulate glycogen synthesis greatly in isolated rat hepatocytes. We extended studies with this compound, designated proglycosyn (Yamaguchi, K., Stephens, T. W., Chikadar, K., Depaoli-Roach, A., And Harris, R. A. (1991) Diabetes 40, (Suppl. 1) 102 (abstr.] employing hepatocytes from normal and streptozotocin diabetic rats. Proglycosyn is more effective than amino acids in stimulating glycogen synthesis. In cells incubated with glucose, lactate, or dihydroxyacetone the effect of glutamine and proglycosyn was synergistic. In cells incubated with glucose plus lactate, or glucose plus dihydroxyacetone, the stimulation by the two agonists was additive. Proglycosyn diverted the gluconeogenic flux from glucose to glycogen. The maximal rates of glycogen deposition attained in the presence of glutamine and proglycosyn from cells incubated with glucose plus lactate, or glucose plus dihydroxyacetone, where about 80 and 110 mumols/h/g of liver, respectively. Proglycosyn depressed glycogenolysis in hepatocytes of fed rats and stimulated glycogen synthesis from lactate and dihydroxyacetone. The incorporation of [U-14C]glucose and [U-14C]lactate in these cells occurred in the presence of glycogen breakdown or exceeded net production, indicating the occurrence of recycling of glycogen in hepatocytes of fed rats. Hepatocytes from fasted streptozotocin diabetic rats contained high levels of glycogen. Glycogenolysis was markedly depressed by proglycosyn. Glycogen synthesis from lactate and dihydroxyacetone in these cells was stimulated by glutamine and proglycosyn in a fashion similar to that in cells from fasted control rats, and the rates of glycogen synthesis were similar in cells of control and diabetic rats. With glucose as sole substrate, glutamine did not stimulate glycogen synthesis. When both agonists were present, there was a marked synergism and substantial glycogen formation. Streptozotocin diabetic rats prior to the onset of cachexia have a normal capacity for glycogen synthesis.

Determination of pathways of glycogen synthesis and the dilution of the three-carbon pool with [U-13C]glucose.

Rats were infused with glucose at 30 mg/min, containing 18% enriched [U-13C]glucose and [1-14C]- and [3-3H]glucose. The mass isotopomer patterns of 13C-labeled blood glucose and liver glycogen were determined by gas chromatography/mass spectroscopy. The contribution of the direct pathway to glycogen was calculated from the three tracers, and the values by all three were nearly identical, about 50%. The 14C specific activity in carbon 6 of glycogen glucose was about 6% that of carbon 1. The [3H]glucose/[1-14C]glucose ratio in glycogen was 80-90% that in blood glucose. The enrichment of 13C and the specific activity of 14C in glycogen formed by the indirect path were 20-25% of glycogen formed directly from glucose. The dilution is of two kinds: (i) an exchange of labeled carbon with unlabeled carbon in the tricarboxylic acid cycle and (ii) dilution by unlabeled nonglucose carbon. Methods to calculate the two types of dilution are presented. In control rats the dilution factor by exchange in the tricarboxylic acid cycle is 1.4, and the dilution by unlabeled carbon is 2.5- to 3.0-fold, with the overall dilution about 4-fold. In rats preinjected with glucagon, the dilution through the tricarboxylic acid cycle was unaffected but that by nonglucose carbon was decreased.

Glucose metabolism and recycling by hepatocytes of OB/OB and ob/ob mice.

Hepatocytes were prepared from livers of ob/ob (obese diabetic) mice and their lean (OB/OB) siblings that had been fasted for 24 h. The hepatocytes were incubated with [U-14C, 2-3H]-, [U-14C, 3-3H]-, and [U-14C, 6-3H]glucose at concentrations from 20 to 120 mM. 14C was recovered mainly in CO2, glycogen, and lactate. Tritium was recovered in water and glycogen. The yield in labeled products from [2-3H]glucose ranged from two to three times that from [U-14C]glucose. The yields from [3-3H]- and [6-3H]glucose were similar, and 1.3-1.7 times that from [U-14C]glucose. At 40 mM, total utilization of glucose by obese mice was about twice that for lean mice, but there was little difference at 120 mM. The rate of recycling between glucose and glucose 6-phosphate was calculated. An equation to calculate the rate of recycling of glucose from the 2-3H/U-14C ratio in glycogen is derived in the APPENDIX. Our results show that 1) the utilization of glucose by hepatocytes from obese diabetic mice exceeds that of their lean controls, 2) the rate of glucose phosphorylation in both groups greatly exceeds glucose uptake and the rate of glycogen synthesis, 3) glucose phosphorylation represents a difference between a high glucokinase rate and hydrolysis of glucose 6-phosphate, and 4) recycling of glucose carbon between glucose 6-phosphate and pyruvate occurs within mouse hepatocytes.

Studies of glycogen synthesis and the Krebs cycle by mass isotopomer analysis with [U-13C]glucose in rats.

Starved rats were infused intragastrically via indwelling duodenal cannulae with glucose at a rate of 30 mg/min/kg. The infusate contained [U-13C]glucose at an enrichment of 32 or 17%. At the end of the infusion, after 160 min, glucose and lactate were isolated from arterial and portal blood and from liver, and liver glycogen was isolated and hydrolyzed to glucose. The enrichment in glucose and lactate and the isotopomer distribution in glucose of masses from 180 to 186 were determined by gas chromatography-mass spectrometry (GC-MS). From analysis of these data we determined (a) gluconeogenesis proceeds at half the basal rate in the presence of a large infused glucose load, (b) one-quarter of the hepatic pyruvate pool is derived from nonglucose carbon, (c) half of the labeled molecules in liver glycogen are of mass 186 from the infused glucose and half are of masses 181-183, (d) the contribution of the indirect path from pyruvate when corrected for synthesis from unlabeled pyruvate ranges from 55 to 65%, (e) the rate of pyruvate carboxylase averages 90% that of citrate synthase, and (f) the rate of exchange of oxaloacetate with fumarate is about three times the rate of flux in the Krebs cycle (four times in the "forward" direction), and the enrichment in carbon 1 of oxaloacetate was 2.3 times that in carbon 4. In the Appendix a method to obtain the isotopomer distribution of newly formed glucose and glycogen glucose is described. An algorithm to correct for the contribution of natural abundance of 13C and the presence of 12C in commercial [U-13C]glucose is presented. A novel mathematical analysis to obtain the parameters of the Krebs cycle from the isotopomer distribution is developed in the Appendix. Equations to calculate the relative rates of pyruvate carboxylase (y), and the equilibration of oxaloacetate with fumarate from the isotopomer distribution are derived. Mass isotopomer analysis provides a novel and powerful tool for the study of carbohydrate metabolism and the operation of the Krebs cycle.

The zonation of liver and the distribution of fructose 2,6-bisphosphate in rat liver.

Livers of starved rats refed for 2 h were perfused in situ by a modification of the dual digitonin pulse technique of Quistorff and Grunnet (Quistorff, B., and Grunnet, N. (1987) Biochem. J. 243, 87-95). A pulse of digitonin (2 mg/ml) was infused first antegrade through the portal vein followed retrograde through the vena cava, or in reverse order, 13 mg of digitonin per zone. Microscopic examination showed that this procedure permeabilized the periportal and perivenous zones of the liver without overlap, with a narrow unaffected band of hepatocytes between the zones. The distribution pattern between periportal and perivenous zones ratio for alanine transaminase, lactate hydrogenase, fructose-1,6-bisphosphatase, and phosphoenolpyruvate carboxykinase ranged from 1.5 to 3. Glucokinase activity was higher in the perivenous zone (periportal/perivenous ratio of 0.7) and glutamine synthetase was exclusively present in that zone. Fructose 2,6-bisphosphate concentration was nearly equal in the two zones.

The role of ATP in the cytostructure of the hepatocytes.

We have previously described the preparation of hepatocytes from which the plasma membrane was removed by digitonin treatment. Such "nude" cells were found to be very stable in sucrose media containing above 50 mM NaCl or KCl, but they disintegrate near instantly in salt-free media, liberating nuclei, mitochondria, and other organelles. We show here that disintegration occurs at a physiologic pH and in the presence of oxygen. Disintegration was blocked by rotenone, oligomycin, KCN, and carboxyatractyloside, establishing that oxidative phosphorylation and ATP generation is essential for disintegration to occur. The addition of ATP, GTP, ITP, or ADP (but not AMP) in the presence of the inhibitors, induced breakdown. Taxol, an inhibitor of tubulin depolymerization and phalloidin, a drug that stabilizes actin fibers, prevented disintegration in salt-free media. The effect of these drugs was counteracted by the addition of ATP. Our results show that two conditions are essential to induce the disintegration of the nude cell: media of low ionic strength, and ATP generation. The ATP effect is likely to be of physiological significance, suggesting role of ATP generation in affecting polymerization of cytoskeletal elements.

Studies with digitonin-treated rat hepatocytes (nude cells).

Isolated rat hepatocytes were treated with digitonin to strip the plasma membrane. The effect of digitonin concentration and exposure time on the recovery of marker enzymes for cell organelles was examined. Hepatocytes treated at room temperature for 1-2 min with 1 mg/ml of digitonin lose some 40% of their protein but retain over 95% of their intact mitochondria and peroxisomes, 90-95% of their endoplasmic reticulum, and about 80% of their lysosomal enzymes. There is little loss of the mitochondrial intermembrane content, and both oxygen uptake and phosphorylation are unimpaired by the treatment. Electron microscopy reveals a complete loss of the plasma membrane, in spite of limited loss of marker enzymes for this membrane. Scanning electron microscopy revealed the interior of the cells to be made up of a dense network of fibers and lamellae attached to the nucleus, mitochondria, and small organelles. The treated cells were stable for many hours when kept in 0.25 M sucrose containing 25 mM monovalent salts. In salt-free sucrose the cells broke up very rapidly into nuclei and other single organelles. Addition of 5 mM NaCl or KCl retards breakup, and 15-20 min were required for dissolution. Intermediate stages, illustrated by scanning electron micrographs, show structure and chains made up mainly of mitochondria held together by a lamellar network. The rapid breakdown occurred at a pH above 7.5 in an oxygen atmosphere and in the presence of phosphate and apparently is an energy-requiring process. It is slow below a pH of 7.2, and at a pH of 6.8 the treated cells remain completely stable in salt-free sucrose. Our results suggest that endoplastic reticulum is a major component of the cytostructure holding together nuclei and organelles.

Mitochondrial-reticular cytostructure in liver cells.

This study examines the structural relationship of mitochondria and the endoplasmic reticulum in liver. Livers of rat and Japanese quail were homogenized and fractionated in media of 0.25 M-sucrose, either 5mM or 50 mM in sodium Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid], pH 7.4 (2.2 mM or 22 mM in Na respectively), designated here as low- and high-salt media. Three particulate fractions were prepared by sequential centrifugation. A nuclear pellet sedimenting at 300 g was obtained as described by Shore & Tata [(1977) J. Cell Biol. 72, 714-725], and from the resulting supernatant thereof a low-speed pellet (1100-1500 g) and a high-speed pellet (8000-10 000 g) were prepared. In the low-salt medium the yields of mitochondrial matrix enzymes (citrate synthase, glutamate dehydrogenase, ornithine carbamoyltransferase) and their specific activities in the low-speed pellet were over twice those in the high-speed pellet. In the high-salt medium the yield of matrix enzymes was 4-5 times, and the specific activities were up to 3 times, higher in the low-speed pellet than in the high-speed pellet. Oxygen uptake and respiratory control ratio were also much higher in the low-speed pellets in both media. Some 50-65% of the microsomal marker enzyme glucose 6-phosphatase was in the supernatant from the high-speed pellet, and the rest sedimented with the mitochondria. Repeated washing with the high-salt medium removes only a limited amount of reticulum. Washing with salt-free sucrose removes most of the reticulum, but a fraction remains strongly bound to mitochondria. Homogenates from quail and rat liver were fractioned isopycnically on Percoll gradients in either 0.25 M-sucrose or 0.25 M-sucrose/50 mM-sodium Hepes. Up to five particulate bands were separated and assayed. Mitochondria were present in two to three bands and were associated with endoplasmic reticulum. As seen in the phase-contrast microscope the mitochondria prepared in the low-salt medium consist of separate organelles. In the high-salt medium the mitochondria appear as chains of from three to ten organelles not touching each other. On addition of univalent ions at concentrations above 20 mM, the mitochondria aggregate into chains, and at higher ionic strength larger multidimensional aggregates are formed. The dispersion and aggregation of mitochondria are reversible. Negatively stained electron micrographs reveal a branched mitochondrial structure, with mitochondria held together by strands of reticulum.(ABSTRACT TRUNCATED AT 400 WORDS)

Glycogen synthesis by hepatocytes from diabetic rats.

Hepatocytes prepared from streptozotocin- and alloxan-diabetic rats starved for 24 h contain 0.5--2% wet wt. of glycogen. Glycogen synthesis in the hepatocytes from such rats, after prior depletion of the glycogen by glucagon injection, was studied. As distinct from cells from normal animals, there was no glycogen synthesis from glucose as sole substrate, even at concentrations of 60 mM. When supplied with glucose, a gluconeogenic precursor (lactate, dihydroxyacetone or fructose), and with glutamine there was concurrent synthesis of glucose and of glycogen. Without glutamine there was little or no glycogen synthesis. The rate of glycogen formation was in the same range as for cells from control rats. Glutamine addition markedly activated glycogen synthase in cells of starved diabetic rats, but there was no effect on phosphorylase. We obtained very little synthesis of glycogen with hepatocytes from fed diabetic rats, whereas with normal animals, synthesis by such cells equals or exceeds that obtained from starved rats. The conversion of synthase b (inactive) into the active form was studied in rat liver homogenates. The activation of the synthase in cells from starved diabetic rats is somewhat less than that from normal animals, but that from fed diabetic rats is markedly decreased compared with that in livers of fed control animals or that of starved diabetic animals.

Glycogen synthesis by rat hepatocytes.

1. Hepatocytes from starved rats or fed rats whose glycogen content was previously depleted by phlorrhizin or by glucagon injections, form glycogen at rapid rates when incubated with 10mM-glucose, gluconeogenic precursors (lactate, glycerol, fructose etc.) and glutamine. There is a net synthesis of glucose and glycogen. 14C from all three types of substrate is incorporated into glycogen, but the incorporation from glucose represents exchange of carbon atoms, rather than net incorporation. 14C incorporation does not serve to measure net glycogen synthesis from any one substrate. 2. With glucose as sole substrate net glucose uptake and glycogen deposition commences at concentrations of about 12--15mM. Glycogen synthesis increases with glucose concentrations attaining maximal values at 50--60mM, when it is similar to that obtained in the presence of 10mM glucose and lactate plus glutamine. 3. The activities of the active (a) and total (a+b) forms of glycogen synthase and phosphorylase were monitored concomitant with glycogen synthesis. Total synthase was not constant during a 1 h incubation period. Total and active synthase activity increased in parallel with glycogen synthesis. 4. Glycogen phosphorylase was assayed in two directions, by conversion of glycose 1-phosphate into glycogen and by the phosphorylation of glycogen. Total phosphorylase was assyed in the presence of AMP or after conversion into the phosphorylated form by phosphorylase kinase. Results obtained by the various methods were compared. Although the rates measured by the procedures differ, the pattern of change during incubation was much the same. Total phosphorylase was not constant. 5. The amounts of active and total phosphorylase were highest in the washed cell pellet. Incubation in an oxygenated medium, with or without substrates, caused a prompt and pronounced decline in the assayed amounts of active and total enzyme. There was no correlation between phosphorylase activity and glycogen synthesis from gluconeogenic substrates. With fructose, active and total phosphorylase activities increased during glycogen syntheses. 6. In glycogen synthesis from glucose as sole substrate there was a decline in phosphorylase activities with increased glucose concentration and increased rates of glycogen deposition. The decrease was marked in cells from fed rats. 7. To determine whether phosphorolysis and glycogen synthesis occur concurrently, glycogen was prelabelled with [2-3H,1-14C]-galactose. During subsequent glycogen deposition there was no loss of activity from glycogen in spite of high amounts of assayable active phosphorylase.

Stimulation of hepatic glycogen synthesis by amino acids.

Hepatocytes isolated from livers of fasted rats form little glycogen from glucose or lactate at concentrations below 20 mM. Glycogen is formed in substantial quantities at a glucose concentration of 60 mM. In the presence of 10 mM glucose, 20-30% as much glycogen as glucose is formed from fructose, sorbitol, or dihydroxyacetone. The addition of either glutamine, alanine, or asparagine stimulates the formation of glycogen from lactate 10- to 40-fold. The formation of glucose and glycogen is then about equal, and glycogen deposition in hepatocytes is similar to rates attained in vivo after fasted rats are refed. The amino acids stimulate 1.5- to 2-fold glycogen synthesis from fructose, and 2- to 4-fold synthesis from dihyDROXYACETONE. Ammonium chloride is about one-half as effective as amino acids in stimulating glycogen synthesis when glucose with lactate are substrates. It increased glycogen synthesis 25-50% from fructose but inhibited synthesis from dihydroxyacetone plus glucose.