Albert Yu

Effects of Arachidonic Acid on Glutamate and γ‐Aminobutyric Acid Uptake in Primary Cultures of Rat Cerebral Cortical Astrocytes and Neurons

Abstract The effects of arachidonic acid on glutamate and γ‐aminobutyric acid (GABA) uptake were studied in primary cultures of astrocytes and neurons prepared from rat cerebral cortex. The uptake rates of glutamate and GABA in astrocytic cultures were 10.4 nmol/mg protein/min and 0.125 nmol/mg protein/min, respectively. The uptake rates of glutamate and GABA in neuronal cultures were 3.37 nmol/mg protein/min and 1.53 nmol/mg protein/min. Arachidonic acid inhibited glutamate uptake in both astrocytes and neurons. The inhibitory effect was observed within 10 min of incubation with arachidonic acid and reached approximately 80% within 120 min in both types of culture. The arachidonic acid effect was not only time‐dependent, but also dose‐related. Arachidonic acid, at concentrations of 0.015 and 0.03 μmol/mg protein, significantly inhibited glutamate uptake in neurons, whereas 20 times higher concentrations were required for astrocytes. The effects of arachidonic acid were not as deleterious on GABA uptake as on glutamate uptake in both astrocytes and neurons. In astrocytes, GABA uptake was not affected by any of the doses of arachidonic acid studied (0.015–0.6 μmol/mg protein). In neuronal cultures, GABA uptake was inhibited, but not to the same degree observed with glutamate uptake. Lower doses of arachidonic acid (0.03 and 0.015 μmol/mg protein) did not affect neuronal GABA uptake. Other polyunsaturated fatty acids, such as docosahexaenoic acid, affected amino acid uptake in a manner similar to arachidonic acid in both astrocytes and neurons. However, saturated fatty acids, such as palmitic acid, exerted no such effect. The significance of the arachidonic acid‐induced inhibition of neurotransmitter uptake in cultured brain cells in various pathological states is discussed.

Glutamate increases glycogen content and reduces glucose utilization in primary astrocyte culture

The glycogen content of primary cultured astrocytes was approximately doubled by incubation with 1 mM L‐glutamate or L‐aspartate. Other amino acids and excitatory neurotransmitters were without effect. The increase in glycogen level was not blocked by the glutamate receptor antagonist kynurenic acid but was completely blocked by the glutamate uptake inhibitor threo‐3‐hydroxy‐D,L‐aspartate and by removal of Na+ from the medium. Incubation with radiolabeled glucose and glutamate revealed that the increased glycogen content was derived almost entirely from glucose. Glutamate at 1 mM was also found to cause a 53 ± 12% decrease in glucose utilization and a 112 ± 69% increase in glucose‐6‐phosphate levels. These results suggest that the glycogen content of astrocytes is linked to the rate of glucose utilization and that glucose utilization can, in turn, be affected by the availability of alternative metabolic substrates. These relationships suggest a mechanism by which brain glycogen accumulation occurs during decreased neuronal activity.

Induction of Intracellular Superoxide Radical Formation by Arachidonic Acid and by Polyunsaturated Fatty Acids in Primary Astrocytic Cultures

Abstract: The effects of arachidonic acid and other polyunsaturated fatty acids (PUFAs) on both oxidative and metabolic perturbation were studied in primary cultures of rat cerebral cortical astrocytes. In the presence of 0.1 mM arachidonic acid, the rate of the reduction of nitroblue tetrazoiium (NBT) to nitroblue formazan (NBF) was stimulated from 0.65 ± 0.10 to 1.43 ± 0.15 and from 0.092 ± 0.006 to 0.162 ± 0.009 nmol/min/mg protein in intact and broken cell preparations, respectively. The rate of superoxide radical formation, as measured by the superoxide dismutase (SOD)‐inhibitable NBT reduction was 0.042 nmol/mg protein in broken cells and was negligible in intact cells. The latter is due to the impermeability of SOD into the intact cell preparation. NBF formation in intact astrocytes stimulated by arachidonic acid was both time‐ and dosedependent. Other PUFAs, including linoleic acid, linolenic acid, and docosahexaenoic acid, were also effective in stimulating NBF formation in astrocytes, whereas saturated palmitic acid and monounsaturated oleic acid were ineffective. Similar effects of these PUFAs were observed in ma‐londialdehyde formation in cells and lactic acid accumulation in incubation medium. These data indicate that both membrane integrity and cellular metabolism were perturbed by arachidonic acid and by other PUFAs. The sites of superoxide radical formation appeared to be intracellular and may be associated with membrane phospholipid domains, because liposome‐entrapped SOD, which was taken up by intact astrocytes, reduced the level of superoxide radicals and lactic acid content, whereas free SOD was not effective.

Cellular and molecular effects of polyunsaturated fatty acids in brain ischemia and injury

Publisher Summary This chapter explains that free polyenoic fatty acids (PUFAs), especially arachidonic acid and docosahexaenoic acid are rapidly released following ischemia, electroconvulsive seizures, and various pathological insults. Free PUFAs and arachidonic acid in particular, have both physiological and pathological effects on cellular systems. It has been described that free arachidonic acid readily intercalates into the membrane and produces significant changes in the packing of the lipid molecules. PUFA-induced membrane fluidity has been associated with the stimulation of chloride transport in corneal epithelium; it enhanced activities of both membrane-associated adenylate cyclase, and guanylate cyclase. The molecular mechanisms of PUFA-induced cellular edema are studied further in the in vitro cortical slices system. The studies have suggested that PUFAs, especially arachidonic acid, play a key role in membrane damage and the development of edema following ischemia and injury.

Regulation of Glycogen Content in Primary Astrocyte Culture: Effects of Glucose Analogues, Phenobarbital, and Methionine Sulfoximine

Abstract: Compounds known to affect glycogen metabolism in vivo or in cell‐free preparations were used to investigate the regulation of glycogen content in intact astrocytes cultured from newborn rat cortex. Compounds were added with fresh medium to culture dishes, and astrocyte glucose and glycogen content determined 24 h later. Increasing the medium glucose concentration from 7.5 mM to 30 mM increased cell glycogen content 80%. Addition of 2‐deoxyglucose or 3‐O‐methyl glucose (2.5–10 mM) also increased cell glycogen content, 50–100%, suggesting a regulatory rather than mass action effect of glucose on astrocyte glycogen content. The phosphorylase b inhibitors 2,2′,4,4′,5,5′‐hexabromobiphenyl and riboflavin had no effect on astrocyte glycogen content, consistent with negligible phosphorylase b activity in normal astrocytes. Phenobarbital and l‐methionine‐DL‐sulfoximine (MSO) are both known to induce astrocyte glycogen accumulation in vivo. The addition of phenobarbital (2 mM) had no effect on the glycogen content of cultured astrocytes, suggesting an indirect mechanism for the in vivo effect. MSO at 1 mM, however, induced a 300% increase in glycogen content. The time course of glucose and glycogen content after MSO administration suggests this increase to be the result of slowed glycogenolysis rather than accelerated glycogen synthesis.

Fructose-1,6-Bisphosphate Protects Astrocytes from Hypoxic Damage

To determine the effects of glucose and fructose-1,6-bisphosphate (FDP) on hypoxic cell damage, primary cultures of astrocytes were incubated for 18 h in an air-tight chamber that had been flushed with 95% N2/5% CO2 for 15 min before it was sealed. Cultures containing 7.5 mM glucose without FDP or FDP without glucose showed evidence of significant cell injury after 18 h of hypoxia (increased lactate dehydrogenase content in the culture medium; cell edema and disruption by phase-contrast microscopy). Cultures exposed to glucose + FDP had normal lactate dehydrogenase concentrations and appeared normal microscopically. Maximal protection of hypoxic cells occurred at 6.0 mM FDP. Lactate concentrations of the culture medium of hypoxic cells increased 2.5 times above normoxic control values when glucose was present, but neither FDP alone nor glucose + FDP caused the lactate concentrations to increase further. This implies that anaerobic glycolysis was not increased by adding FDP to the medium. Cell volumes (water space) measured with [14C]-3-0-methyl-D-glucose were normal with glucose + FDP in the culture medium of hypoxic cells but were significantly larger than normal when glucose alone was present. Increases in cell volume paralleled changes in lactate dehydrogenase in the culture medium. Uptake of [14C]FDP occurred rapidly in normoxic cells and was maximal after 5 min of incubation. The data indicate that the presence of glucose + FDP in the culture medium protects primary cultures of hypoxic astrocytes from cell damage.

Fructose-1,6-bisphosphate reduces ATP loss from hypoxic astrocytes

Hypoxia caused injury and metabolic dysfunction of astrocytes, as indicated by a time-dependent loss of lactate dehydrogenase (LDH) activity and ATP content. The combination of 3.5 mM fructose-1,6-bisphosphate (FBP) and 7.5 mM glucose (GLC) reduced the decrease of ATP and prevented the loss of LDH. These data indicate that the combination of GLC + FBP protects astrocytes from hypoxia. The results also suggest that the maintainance of ATP concentration is the mechanism by which FBP prevents hypoxic injury.

Hepatocellular carcinoma screening practices and impact on survival among hepatitis B-infected Asian Americans.

Summary.  Asians Americans have a high burden of hepatitis B virus (HBV) associated hepatocellular carcinoma (HCC). HCC screening practices in this population are unknown. We aimed to investigate predictors and patterns of HCC screening and its impact on survival in HBV‐infected Asian Americans. Clinical data were obtained from a retrospective cohort of 1870 HBsAg‐positive Asians in San Francisco’s safety net clinics. In 824 patients at‐risk for HCC, screening (≥1 imaging and/or AFP per year) decreased from 67% to 47% to 24% from the 1st to 2nd to 10th year after HBV diagnosis, respectively. AFP, imaging, and imaging plus AFP were used in 37%, 14%, and 49% during the first year after diagnosis, and imaging plus AFP increased to 64% by the 10th year. Among 1431 patients followed in 2007, age 40–64 years, female gender, cirrhosis, hepatologist evaluation, HBV diagnosis after 2003, and testing for HBeAg were associated with HCC screening. Of the 51 patients with HCC, more cirrhotics received screening and were diagnosed with early stage disease. Median survival following HCC diagnosis was higher in screened patients (1624 days vs. 111 days, P = 0.02). MELD score at HCC diagnosis (HR 1.2, 95% CI 1.1–1.3) and receipt of curative therapy (HR 0.3, 95% CI 0.08–0.94) were associated with survival. Screening rates in at‐risk Asian Americans, particularly among noncirrhotics, were suboptimal and decreased over time. Among patients with HCC, receipt of prior screening improved survival, and this survival benefit was related to better liver function at HCC diagnosis and receipt of curative therapy.

Chapter 21: Glutamate as an energy substrate for neuronal-astrocytic interactions

Publisher Summary This chapter discusses the role of glutamate as a metabolic substrate. Glutamate is the most plentiful amino acid and the major excitatory neurotransmitter in adult central nervous system (CNS). Glutamate participates in the synthesis of proteins, peptides and fatty acids, and in the control of osmotic or anionic balance. It is a constituent of at least two important co-factors, glutathione and folic acid; it contributes along with glutamine to the regulation of ammonia levels, and it serves as precursor for GABA and various tricarboxylic acid (TCA) cycle intermediates. Glutamate is released from neurons in large amounts. Uptake studies demonstrates that both neurons and astrocytes take up glutamate. The uptake of glutamate into astrocytes represents a net transfer of carbon skeleton from the neurons to astrocytes. There are three probable roles for this uptake process: (1) to remove the glutamate from extracellular space and synaptic clefts as a means of termination of the transmitter activity; (2) to form glutamine during the detoxification of ammonia and (3) to serve as a metabolic substrate for astrocytes. Metabolic studies have shown that, a part of the glutamate taken up by astrocytes is metabolized to CO2 and another part to glutamine, the latter of which then can be returned to neurons as a precursor for glutamate and GABA. Glutamate exerts a regulatory effect on glycogen metabolism in astrocytes and also affects glucose utilization in astrocytes. Glutamate also plays an important role in the pathogenesis of various neurologic diseases and insult.

Provider, Patient, and Practice Factors Shape Hepatitis B Prevention and Management by Primary Care Providers.

Goals: To evaluate provider knowledge, attitudes and barriers to hepatitis B virus (HBV) care and management practices across diverse primary care settings. Background: Factors influencing adherence to recommended HBV screening and management guidelines are poorly defined. Materials and Methods: Providers across various health care settings in San Francisco were surveyed. Multivariate analyses were used to identify factors associated with recommended HBV screening, vaccination, and disease monitoring. Results: Of 277 (41.3%) responding providers, 42% reported performing HBV screening in >50% of at-risk patients, and 49%, HBV vaccination in >50% of eligible patients. Most reported appropriate monitoring of a majority of HBV-infected patients with alanine aminotransferase (79%) and HBV viral load (67%) every 6 to 12 months, but performed any hepatocellular carcinoma screening in 49%. Provider factors significantly associated with HBV screening were speaking an Asian language [odds ratio (OR), 3.27], offering HBV treatment (OR, 3.00), having >25% of Asian patients in practice (OR, 2.10), practicing in safety net settings (OR, 7.51) and having higher barrier score (OR, 0.74). Appropriate HBV monitoring was associated with provider speaking an Asian language (OR, 3.43) and provider age (OR, 0.68/decade). Hepatocellular carcinoma screening was associated with having >25% of patients speaking English as a second language (OR, 4.26) and practicing in safety net settings (OR, 0.14). Conclusions: Rates of adherence to HBV guidelines were suboptimal irrespective of practice setting and were influenced by certain provider, patient and practice factors. This study reinforces the importance of engaging primary care providers in development, dissemination, and implementation of evidence-based HBV practice guidelines.