Acss2 Deletion Reveals Functional Versatility via Tissue-Specific Roles in Transcriptional Regulation.

The coordination of cellular biological processes is regulated in part via metabolic enzymes acting to match cellular metabolism to current conditions. The acetate activating enzyme, acyl-coenzyme A synthetase short-chain family member 2 (Acss2), has long been considered to have a predominantly lipogenic function. More recent evidence suggests that this enzyme has regulatory functions in addition to its role in providing acetyl-CoA for lipid synthesis. We used Acss2 knockout mice (Acss2-/-) to further investigate the roles this enzyme plays in three physiologically distinct organ systems that make extensive use of lipid synthesis and storage, including the liver, brain, and adipose tissue. We examined the resulting transcriptomic changes resulting from Acss2 deletion and assessed these changes in relation to fatty acid constitution. We find that loss of Acss2 leads to dysregulation of numerous canonical signaling pathways, upstream transcriptional regulatory molecules, cellular processes, and biological functions, which were distinct in the liver, brain, and mesenteric adipose tissues. The detected organ-specific transcriptional regulatory patterns reflect the complementary functional roles of these organ systems within the context of systemic physiology. While alterations in transcriptional states were evident, the loss of Acss2 resulted in few changes in fatty acid constitution in all three organ systems. Overall, we demonstrate that Acss2 loss institutes organ-specific transcriptional regulatory patterns reflecting the complementary functional roles of these organ systems. Collectively, these findings provide further confirmation that Acss2 regulates key transcription factors and pathways under well-fed, non-stressed conditions and acts as a transcriptional regulatory enzyme.

Miasmas, germs, homeopathy and hormesis: commentary on the relationship between homeopathy and hormesis.

Is hormesis related to homeopathy? Despite the superficial similarity of the low dose of the applied stimulus, there are compelling reasons for maintaining hormesis and homeopathy as unrelated. Homeopathy originated in the medical knowledge vacuum of the 19th century, prior to the acceptance of the germ/gene bases of disease. Homeopathy was never grounded on empirical scientific evidence. Hormesis, on the other hand, has always been an empirical science, involving properly controlled experiments. Hormesis is a concept in toxicology that involves biphasic dose responses in biological systems, wherein low doses of stressors can have beneficial effects and higher doses have harmful effects. Hormesis, as it applies to toxicology, is a necessary and useful concept describing adaptive organismic responses to applied stressors. Conversely, homeopathy is a medical doctrine based on the erroneous belief that substances which cause the symptoms of a disorder will cure the disorder when given to patients in small doses. To suggest that homeopathy is a form of post-exposure conditioning hormesis assumes that homeopathic practitioners employed the scientific method with measurable experimental end-points and proper controls, and that their 'provings' had actually determined the correct compound, at the correct dose, required to cure a disorder. Because many homeopathic preparations are diluted to a point where none of the starting solutes would likely remain, the idea of a beneficial or harmful hormetic dose becomes moot. Without supporting scientific evidence for the efficacy or purported mechanisms of homeopathy, the term hormesis should not be linked with it in any way.

Metabolic acetate therapy improves phenotype in the tremor rat model of Canavan disease.

Genetic mutations that severely diminish the activity of aspartoacylase (ASPA) result in the fatal brain dysmyelinating disorder, Canavan disease. There is no effective treatment. ASPA produces free acetate from the concentrated brain metabolite, N-acetylaspartate (NAA). Because acetyl coenzyme A is a key building block for lipid synthesis, we postulated that the inability to catabolize NAA leads to a brain acetate deficiency during a critical period of CNS development, impairing myelination and possibly other aspects of brain development. We tested the hypothesis that acetate supplementation during postnatal myelination would ameliorate the severe phenotype associated with ASPA deficiency using the tremor rat model of Canavan disease. Glyceryltriacetate (GTA) was administered orally to tremor rats starting 7 days after birth, and was continued in food and water after weaning. Motor function, myelin lipids, and brain vacuolation were analyzed in GTA-treated and untreated tremor rats. Significant improvements were observed in motor performance and myelin galactocerebroside content in tremor rats treated with GTA. Further, brain vacuolation was modestly reduced, and these reductions were positively correlated with improved motor performance. We also examined the expression of the acetyl coenzyme A synthesizing enzyme acetyl coenzyme A synthase 1 and found upregulation of expression in tremor rats, with a return to near normal expression levels in GTA-treated tremor rats. These results confirm the critical role played by NAA-derived acetate in brain myelination and development, and demonstrate the potential usefulness of acetate therapy for the treatment of Canavan disease.

Enzyme stabilization by glass-derived silicates in glass-exposed aqueous solutions.

OBJECTIVES: To analyze the solutes leaching from glass containers into aqueous solutions, and to show that these solutes have enzyme activity stabilizing effects in very dilute solutions. METHODS: Enzyme assays with acetylcholine esterase were used to analyze serially succussed and diluted (SSD) solutions prepared in glass and plastic containers. Aqueous SSD preparations starting with various solutes, or water alone, were prepared under several conditions, and tested for their solute content and their ability to affect enzyme stability in dilute solution. RESULTS: We confirm that water acts to dissolve constituents from glass vials, and show that the solutes derived from the glass have effects on enzymes in the resultant solutions. Enzyme assays demonstrated that enzyme stability in purified and deionized water was enhanced in SSD solutions that were prepared in glass containers, but not those prepared in plastic. The increased enzyme stability could be mimicked in a dose-dependent manner by the addition of silicates to the purified, deionized water that enzymes were dissolved in. Elemental analyses of SSD water preparations made in glass vials showed that boron, silicon, and sodium were present at micromolar concentrations. CONCLUSIONS: These results show that silicates and other solutes are present at micromolar levels in all glass-exposed solutions, whether pharmaceutical or homeopathic in nature. Even though silicates are known to have biological activity at higher concentrations, the silicate concentrations we measured in homeopathic preparations were too low to account for any purported in vivo efficacy, but could potentially influence in vitro biological assays reporting homeopathic effects.

Laboratory research in homeopathy: con.

Alternative medical approaches to human diseases such as cancer are becoming increasingly popular, but reports on their success rates have been highly variable. Homeopathy is an alternative medical practice often applied to less critical human diseases but one that has also been applied sporadically to the treatment of cancer. Animal studies on the use of homeopathy to treat experimental cancer are few and the evidence provided to date is far from conclusive. The debate presented here concerns the utility of animal studies on cancer treatment with homeopathic preparations. As part of a Point-Counterpoint feature, this review and its companion piece in this issue by Khuda-Bukhsh (Integr Cancer Ther. 2006;5:320-332) are composed of a thesis section, a response section in reaction to the companion thesis, and a rebuttal section to address issues raised in the companion response.

Canavan disease and the role of N-acetylaspartate in myelin synthesis.

Canavan disease (CD) is an autosomal-recessive neurodegenerative disorder caused by inactivation of the enzyme aspartoacylase (ASPA, EC 3.5.1.15) due to mutations. ASPA releases acetate by deacetylation of N-acetylaspartate (NAA), a highly abundant amino acid derivative in the central nervous system. CD results in spongiform degeneration of the brain and severe psychomotor retardation, and the affected children usually die by the age of 10. The pathogenesis of CD remains a matter of inquiry. Our hypothesis is that ASPA actively participates in myelin synthesis by providing NAA-derived acetate for acetyl CoA synthesis, which in turn is used for synthesis of the lipid portion of myelin. Consequently, CD results from defective myelin synthesis due to a deficiency in the supply of the NAA-derived acetate. The demonstration of the selective localization of ASPA in oligodendrocytes in the central nervous system (CNS) is consistent with the acetate deficiency hypothesis of CD. We have tested this hypothesis by determining acetate levels and studying myelin lipid synthesis in the ASPA gene knockout model of CD, and the results provided the first direct evidence in support of this hypothesis. Acetate supplementation therapy is proposed as a simple and inexpensive therapeutic approach to this fatal disease, and progress in our preclinical efforts toward this goal is presented.

Progress toward acetate supplementation therapy for Canavan disease: glyceryl triacetate administration increases acetate, but not N-acetylaspartate, levels in brain.

Canavan disease (CD) is a fatal genetic neurodegenerative disorder caused by mutations in the gene for aspartoacylase, an enzyme that hydrolyzes N-acetylaspartate (NAA) into L-aspartate and acetate. Because aspartoacylase is localized in oligodendrocytes, and NAA-derived acetate is incorporated into myelin lipids, we hypothesize that an acetate deficiency in oligodendrocytes is responsible for the pathology in CD, and we propose acetate supplementation as a possible therapy. In our preclinical efforts toward this goal, we studied the effectiveness of orally administered glyceryl triacetate (GTA) and calcium acetate for increasing acetate levels in the murine brain. The concentrations of brain acetate and NAA were determined simultaneously after intragastric administration of GTA. We found that the acetate levels in brain were increased in a dose- and time-dependent manner, with a 17-fold increase observed at 1 to 2 h in 20- to 21-day-old mice at a dose of 5.8 g/kg GTA. NAA levels in the brain were not significantly increased under these conditions. Studies using mice at varying stages of development showed that the dose of GTA required to maintain similarly elevated acetate levels in the brain increased with age. Also, GTA was significantly more effective as an acetate source than calcium acetate. Chronic administration of GTA up to 25 days of age did not result in any overt pathology in the mice. Based on these results and the current Food and Drug Administration-approved use of GTA as a food additive, we propose that it is a potential candidate for use in acetate supplementation therapy for CD.

Immunohistochemical localization of aspartoacylase in the rat central nervous system.

Aspartoacylase (ASPA; EC 3.5.1.15) catalyzes deacetylation of N-acetylaspartate (NAA) to generate free acetate in the central nervous system (CNS). Mutations in the gene coding ASPA cause Canavan disease (CD), an autosomal recessive neurodegenerative disease that results in death before 10 years of age. The pathogenesis of CD remains unclear. Our working hypothesis is that deficiency in the supply of the NAA-derived acetate leads to inadequate lipid/myelin synthesis during development, resulting in CD. To explore the localization of ASPA in the CNS, we used double-label immunohistochemistry for ASPA and several cell-specific markers. A polyclonal antibody was generated in rabbit against mouse recombinant ASPA, which reacted with a single band (approximately 37 kD) on Western blots of rat brain homogenate. ASPA colocalized throughout the brain with CC1, a marker for oligodendrocytes, with 92-98% of CC1-positive cells also reactive with the ASPA antibody. Many cells were labeled with ASPA antibodies in white matter, including cells in the corpus callosum and cerebellar white matter. Relatively fewer cells were labeled in gray matter, including cerebral cortex. No astrocytes were labeled for ASPA. Neurons were unstained in the forebrain, although small numbers of large reticular and motor neurons were faintly to moderately stained in the brainstem and spinal cord. Many ascending and descending neuronal fibers were moderately stained for ASPA in the medulla and spinal cord. Microglial-like cells showed faint to moderate staining with the ASPA antibodies throughout the brain by the avidin/biotin-peroxidase detection method, and colocalization studies with labeled lectins confirmed their identity as microglia. The predominant immunoreactivity in oligodendrocytes is consistent with the proposed role of ASPA in myelination, supporting the case for acetate supplementation as an immediate and inexpensive therapy for infants diagnosed with CD.