A␤ 1-42 is a self-associating peptide whose neurotoxic derivatives are thought to play a role in Alzheimer's pathogenesis. Neurotoxicity of amyloid ␤ protein (A␤) has been attributed to its fibrillar forms, but experiments presented here characterize neurotoxins that assemble when fibril formation is inhibited. These neurotoxins comprise small diffusible A␤ oligomers (referred to as ADDLs, for A␤-derived diffusible ligands), which were found to kill mature neurons in organotypic central nervous system cultures at nanomolar concentrations. At cell surfaces, ADDLs bound to trypsin-sensitive sites and surface-derived tryptic peptides blocked binding and afforded neuroprotection. Germ-line knockout of Fyn, a protein tyrosine kinase linked to apoptosis and elevated in Alzheimer's disease, also was neuroprotective. Remarkably, neurological dysfunction evoked by ADDLs occurred well in advance of cellular degeneration. Without lag, and despite retention of evoked action potentials, ADDLs inhibited hippocampal long-term potentiation, indicating an immediate impact on signal transduction. We hypothesize that impaired synaptic plasticity and associated memory dysfunction during early stage Alzheimer's disease and severe cellular degeneration and dementia during end stage could be caused by the biphasic impact of A␤-derived diffusible ligands acting upon particular neural signal transduction pathways.

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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.

1. The metabolism of glucose labeled uniformly with I4C, and in positions 2, 3 and 5 with tritium by hepatocytes from fed and fasted rats were studied. Cells were incubated with glucose as sole substrate, or with glucose and a variety of glucose precursors, and uptake or production of glucose, and the utilization of the isotopes was determined.2. There was no uptake of glucose at concentration of up to 15 mM, and net glucose synthesis in the presence of precursors. I4C was however recovered in COz, lactate and amino acids, and tritium in water. Considerable incorporation into glycogen from '"C and 3H-labeled glucose occurred at high (above 20 mM) glucose concentrations.3. The yield in water always exceeded that in I4C-labeled products. The yield in 3HOH from [2-3H]glucose exceeded that from [5-3H]glucose, and the latter was greater than from [3-3H]glucose.4. Utilization of labeled glucose does not follow Michaelis-Menten kinetics. The fractional rate of uptake of 14C and tritium-labeled glucose increases with glucose concentration with a maximum at about 15 mM and then declines.5. The effect of numerous gluconeogenic substrates on the isotope utilization and the 3H/14C ratio in glycogen was studied. The uptake of I4C was always depressed. Addition of lactate and dihydroxyacetone has little effect on the detritiation of [2-3H] 6. Equations to calculate the phosphorylation of glucose and fructose 6-phosphate in the presence of futile cycling between glucose and glucose 6-phosphate and fructose 6-phosphate and fructose 1,6-bisphosphate were derived.7. The estimate of glucose phosphorylation requires determination of the specific activity of glucose 6-phosphate from [2-3H]glucose. It appears that futile cycling between glucose and glucose 6-phosphate is extensive in cells with a high glycogen content, but is low in cells from starved rats and nearly absent in those from diabetic animals.8. The estimation of the phosphorylation of fructose 6-phosphate in the presence of cycling requires knowledge of the specific activities of fructose 6-phosphate and fructose 1,6-bisphosphate from [3-3H]glucose. At present there are no adequate data to calculate phosphorylation and recycling of fructose 6-phosphate, but under some conditions the rate may be quite high.Liver contains the full complement of enzymes for glucose synthesis and for glycolysis. Two irreversible steps in glucose metabolism are between glucose and glucose-6-P, and between fructose-6-P and fmctose-1,6-P2. If the kinases and phosphatases catalyzing these conversions were simultaneously active, there would be two futile cycles, namely glucose + glucose-6-P + glucose (the glucose cycle), and fructose-6-P + fructose-l,6-P2 + fructose-6-P (the fructose 6-P cycle). Newsholme and coworkers [l -31 have sugAbbreviations. Glucose-6-P, glucose 6-phosphate; fructose-6-P, fructose 6-phosphate; fructose 1,6-P,, fructose 1,6-bisphosphate; glyceraldehyde-3-P, glyceraldehyde 3-phosphate.gested that futile cycles have a role in metabolic regulation. The fructose-6-P cycle serve...

Elevated expression of glial fibrillary acidic protein (GFAP) is associated with astrocyte activation during responses to injury in the CNS. Because transforming growth factor-␤1 (TGF-␤1) and interleukin-1␤ (IL-1␤) are released during neural responses to injury and because these cytokines also modulate GFAP mRNA levels, it is of interest to define their role in GFAP transcription. The increases of GFAP mRNA in response to TGF-␤1 and decreases in response to IL-1␤ were shown to be transcriptionally mediated in rat astrocytes transfected with a luciferase-reporter construct containing 1.9 kb of 5Ј-upstream rat genomic DNA. Constructs containing sequential deletions of the rat GFAP 5Ј-upstream promoter identified a short region proximal to the transcription start (Ϫ106 to Ϫ53 bp) that provides full responses to TGF-␤1 and IL-1␤. This region contains an unusual sequence motif with overlapping nuclear factor-1 (NF-1)-and nuclear factor-B (NF-B)-like binding sites and homology to known TGF-␤ response elements. Mutagenesis (3-bp exchanges) in Ϫ70 to Ϫ68 bp blocked the induction of GFAP by TGF-␤1 and the repression by IL-1␤. Gel shift experiments showed that the DNA segment Ϫ85 to Ϫ63 bp was bound by a factor(s) in nuclear extracts from astrocytes. The concentrations of these DNA binding factors were increased by treatment of astrocytes with TGF-␤1 and decreased by IL-1␤. Binding of these nuclear factors was blocked by mutation of Ϫ70 to Ϫ68 bp. Despite homology to NF-1 or NF-B binding sites in the GFAP promoter at segment Ϫ79 to Ϫ67 bp, anti-NF-B or anti-NF-1 antibodies did not further retard the gel shift of the nuclear factors/DNA complex. Moreover, astrocytic nuclear proteins do not compete for the specific binding to NF-1 consensus sequence. Thus, nuclear factors from astrocytes that bind to the Ϫ85-to Ϫ63-bp promoter segment might be only distantly related to NF-1 or NF-B. These findings are pertinent to the use of GFAP promoter constructs in transgenic animals, because cisacting elements in the GFAP promoter are sensitive to cytokines that may be elaborated in response to expression of transgene products. Key Words: Glial fibrillary acidic protein promoter-Transforming growth factor-␤1-Interleukin-1␤-Nuclear factor-1 binding site.

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. Isolated rat hepatocytes form glucose from numerous substrates at a rate similar to that in vivo, and such cells have served in recent years as a major preparation for the study of gluconeogenesis. However, attempts to obtain hepatocytes capable of efficient glycogen synthesis have not been successful. We set out to develop a hepatocyte preparation capable of synthesis, from common gluconeogenic substrates at moderate concentrations, similar to that obtained in vivo (1-2 ,tmol of glucose equivalents per min/g) within 1 hr after refeeding fasted rats. We report here that certain amino acids stimulate glycogen formation from lactate to this extent. Our findings suggest that certain amino acids or their derivatives may have a major role in the regulation of glycogen synthesis. METHODSMale rats of the Sprague-Dawley strain which weighed 180-220 g were fasted for 24 hr. The rats were used for hepatocyte preparation without further treatment, or they were injected intraperitoneally with a solution, 1 ml/100 g of body weight, of either 5% glucose plus 5% fructose, or 1 mg/100 g of body weight of prednisolone succinate. They were anesthesized with nembutal and operated on 30 min after the injection of sugar and 1 hr after that of prednisolone.Hepatocytes were prepared as previously described (1) except that the bile duct and adjacent vein were not ligated prior to cannulation of the portal vein. The yield from a 200 g rat was 6-8 ml of washed and lightly packed cells. Between 0.04 and 0.06 ml of cells were incubated in 3 ml of Krebs bicarbonate buffer, usually for 1 hr, in an atmosphere of 95% 02-5% CO2, at 380. Incubations were terminated by injecting perchloric acid to a 3% final concentration. Each substrate combination was incubated in duplicate, and results between them nearly always agreed to within 10%. Enzymatic procedures were used to determine glucose, fructose and lactate (1), urea and ammonia (2), glutamate (3), glutamine (4), asparagine and aspartate (5), and alanine (6) spectrophotometrically. Glycogen was determined acco...

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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.