Mechanism of activation of choline acetyltransferase in a human neuroblastoma cell line

In our previous report we have shown that the enzyme choline acetyltransferase (ChAT), responsible for the synthesis of the neurotransmitter acetylcholine, can be regulated in response to treatment by either retinoic acid or sodium butyrate. These responses were dose and time dependent, but the mechanism by which these agents were acting was not understood. We now report the results of studies aimed at elucidating the level at which both sodium butyrate and retinoic acid are able to increase ChAT activity. The effects of these agents on macromolecular synthesis appeared to be limited to small but statistically significant increases in the rate of RNA synthesis. However, inhibition of DNA, RNA and protein synthesis in these cells had no effect on the stimulation of ChAT activity by either sodium butyrate or retinoic acid. Several experiments appeared to rule out a role for cyclic AMP or protein kinase C in the regulation of ChAT activity, even though retinoic acid treatment could increase endogenous levels of cyclic AMP 3- to 4-fold over the time course of ChAT activity stimulation. Experiments performed to determine kinetic parameters of this enzyme demonstrated changes only in the V_max, but not the K_m of ChAT, suggesting that the affinity of enzyme for either of its substrates was not responsible for the increase in specific activity. Taken together, this evidence suggests that the activation of choline acetyltransferase in this human neuroblastoma cell line occurs at the post-translational level.     

The Influence of Specific Luminal Factors on the Colonic Epithelium: High-Dose Butyrate and Physical Changes Suppress Early Carcinogenic Events in Rats

INTRODUCTION Although luminal delivery of butyrate is one putative mechanism by which biology of the colonic epithelium might be influenced by changes in luminal contents, there is a paucity of supportive cause–effect evidence. This study aimed to directly establish whether distal colonic butyrate delivery is able to alter the response of the distal colonic epithelium to a carcinogen.METHODS Groups of male Sprague-Dawley rats with chronically intubated colons received infusions of 80 mM butyrate or 0.9 percent saline into distal colon two or five times daily. Three weeks after exposure to azoxymethane (15 mg/kg subcutaneously), the density of aberrant crypts was quantified in distal colon.RESULTS Infusions of 0.5 ml twice daily, whether containing saline or butyrate, decreased the number of aberrant crypt foci by 45 percent compared with rats receiving no infusions (P = 0.004, analysis of variance). Similar results were obtained when infusions were restricted to the postinitiation phase. When infusions were increased to 1 ml five times daily, saline infusions similarly suppressed aberrant crypt formation (38 percent), but butyrate infusions suppressed it to a greater degree (by 64 percent; P = 0.02 compared with saline infusion, t-test).CONCLUSIONS High levels of butyrate delivery to the distal colonic lumen alter the epithelial response to a carcinogen in otherwise healthy rats. This finding directly supports the notion that the effects of butyrate on cells in vitro do occur in vivo provided a sufficient dose is delivered. The effect of infusion of liquid per se on the epithelial response highlights the potential impact physical changes alone can have on the colonic epithelium.     

Role of short chain fatty acids in gut health and possible therapeutic approaches in inflammatory bowel diseases

Inflammatory bowel diseases (IBDs) are characterized by inflammation in the gastrointestinal tract and include Ulcerative Colitis and Crohn’s Disease. These diseases are costly to health services, substantially reduce patients’ quality of life, and can lead to complications such as cancer and even death. Symptoms include abdominal pain, stool bleeding, diarrhea, and weight loss. The treatment of these diseases is symptomatic, seeking disease remission. The intestine is colonized by several microorganisms, such as fungi, viruses, and bacteria, which constitute the intestinal microbiota (IM). IM bacteria promotes dietary fibers fermentation and produces short-chain fatty acids (SCFAs) that exert several beneficial effects on intestinal health. SCFAs can bind to G protein-coupled receptors, such as GPR41 and GPR43, promoting improvements in the intestinal barrier, anti-inflammatory, and antioxidant effects. Thus, SCFAs could be a therapeutic tool for IBDs. However, the mechanisms involved in these beneficial effects of SCFAs remain poorly understood. Therefore, this paper aims to provide a review addressing the main aspects of IBDs, and a more detailed sight of SCFAs, focusing on the main effects on different aspects of the intestine with an emphasis on IBDs.     

EXCITING NEW TREATMENT APPROACHES FOR PATHYPHYSIOLOGIC MECHANISMS OF SICKLE CELL DISEASE

During the past several decades, our understanding of the complex pathophysiology of vasoocclusion associated with sickle cell disease has improved greatly. Interaction of genes, hemoglobin molecules, red cell membrane and metabolic changes, cell-cell interactions and cell-plasma interactions, red cell adhesion to vascular endothelium, activation of coagulation, and vascular reactivity play a role in vaso occlusion. Penicillin prophylaxis of pneumococcal infections and appropriate use of blood transfusions and other supportive measures improved survival of sickle cell patients. Hydroxyurea made a major impact on sickle cell therapy when it was shown to decrease acute painful episodes, acute chest syndrome, and the need for blood transfusion in adults. Significant experience in the use of hydroxyurea has been accumulated in older children. The benefits and risks of hydroxyurea for younger children and long-term risks in all patients will be evaluated in future investigations. Other promising therapies include butyrate compounds, clotrimazole, magnesium supplementation, poloxamer 188, antiadhesion agents, anticoagulant approaches, and nitric oxide. Hemopoietic transplantation remains the only curative therapy. However, several transgenic mouse models are available for studies of gene therapy or other treatment approaches on biochemical, cellular, and pathologic effects of mutant genes.     

Improving healthspan via changes in gut microbiota and fermentation

Dietary resistant starch impact on intestinal microbiome and improving healthspan is the topic of this review. In the elderly population, dietary fiber intake is lower than recommended. Dietary resistant starch as a source of fiber produces a profound change in gut microbiota and fermentation in animal models of aging. Dietary resistant starch has the potential for improving healthspan in the elderly through multiple mechanisms as follows: (1) enhancing gut microbiota profile and production of short-chain fatty acids, (2) improving gut barrier function, (3) increasing gut peptides that are important in glucose homeostasis and lipid metabolism, and (4) mimicking many of the effects of caloric restriction including upregulation of genes involved in xenobiotic metabolism.     

Anti-inflammatory properties of the short-chain fatty acids acetate and propionate： A study with relevance to inflammatory bowel disease

AIM: To compare the anti-inflammatory properties of butyrate with two other SCFAs,namely acetate and propionate,which have less well-documented effects on inflammation. METHODS: The effect of SCFAs on cytokine release from human neutrophils was studied with ELISA. SCFA-dependent modulation of NF-κB reporter activity was assessed in the human colon adenocarcinoma cell line,Colo320DM. Finally,the effect of SCFAs on gene expression and cytokine release,measured with RT-PCR and ELISA,respectively,was studied in mouse colon organ cultures established from colitic mice. RESULTS: Acetate,propionate and butyrate at 30 mmol/L decreased LPS-stimulated TNFα release from neutrophils,without affecting IL-8 protein release. All SCFAs dose dependently inhibited NF-κB reporter activity in Colo320DM cells. Propionate dose-dependently suppressed IL-6 mRNA and protein release from colon organ cultures and comparative studies revealed that propionate and butyrate at 30 mmol/L caused a strong inhibition of immune-related gene expression,whereas acetate was less effective. A similar inhibition was achieved with the proteasome inhibitor MG-132,but not the p38 MAPK inhibitor SB203580. All SCFAs decreased IL-6 protein release from organ cultures. CONCLUSION: In the present study propionate and butyrate were equipotent,whereas acetate was less effective,at suppressing NF-κB reporter activity,immune-related gene expression and cytokine release in vitro. Our findings suggest that propionate and acetate,in addition to butyrate,could be useful in the treatment of inflammatory disorders,including IBD.     

Mucosal metabolism in ulcerative colitis and crohn's disease

PURPOSE: Colonic mucosal metabolism of butyrate may be impaired in ulcerative colitis. In this study we sought to confirm this observation, to determine if a similar change occurs in Crohn's colitis, and to establish whether a panenteric disorder of butyrate metabolism exists in either condition. METHODS: With use of a microculture technique, mucosal metabolic fluxes of¹⁴[C]-labeled butyrate and¹⁴[C]-labeled glutamine were measured as¹⁴[C] carbon dioxide production in mucosal biopsy specimens from the colon and ileum in patients with ulcerative colitis, Crohn's colitis, and healthy bowel. Results were expressed as pmol/µg biopsy DNA/hour. RESULTS: In the colon the mucosal metabolic fluxes of both butyrate and glutamine are reduced in both ulcerative colitis and Crohn's colitis compared with healthy controls. These changes were most marked in the presence of moderate to severe mucosal inflammation, there being no significant difference in mucosal metabolic flux between mildly inflamed mucosa and healthy controls. In the ileum the mucosal metabolic fluxes of butyrate and glutamine did not differ between healthy controls and those with either ulcerative colitis or Crohn's colitis. CONCLUSIONS: Changes in colonic mucosal metabolism of butyrate and glutamine in inflammatory bowel disease occur as a consequence of the inflammatory process and are not peculiar to ulcerative colitis. Ileal mucosal metabolism is unchanged in ulcerative colitis and Crohn's colitis, indicating the absence of a panenteric abnormality of mucosal metabolism in these two conditions.Supported by the Mater College, Dublin, Ireland.Portions of this work were read at the American Gastroenterological Association San Francisco, California, May 19 to 24, 1996, and an abstract was published in Gastroenterology 1996;110:A900.     

Upregulation of 25-hydroxyvitamin D3-1α-hydroxylase by butyrate in Caco-2 cells

AIM: To investigate the possible involvement of 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase [1alpha-25(OH) (2) D(3)] in butyrate-induced differentiation in human intestinal cell line Caco-2 cells. METHODS: Caco-2 cells were incubated either with 3 mmol/L butyrate and 1 micromol/L 25(OH) (2) D(3) or with 1 micromol/L 1alpha-25(OH) (2) D(3) for various time intervals ranging from 0 to 72 h. Additionally, cells were co-incubated with butyrate and either 25(OH) (2) D(3) or 1alpha-25(OH) (2) D(3). 1alpha-25(OH) (2) D(3) mRNA was determined semi-quantitatively using the fluorescent dye PicoGreen. Immunoblotting was used for the detection of 1alpha-25(OH) (2) D(3) protein. Finally, enzymatic activity was measured by ELISA. RESULTS: Both butyrate and 1alpha-25(OH) (2) D(3) stimulated differentiation of Caco-2 cells after a 48 h incubation period, while 25(OH) (2) D(3) had no impact on cell differentiation. Synergistic effects on differentiation were observed when cells were co-incubated with butyrate and vitamin D metabolite. Butyrate transiently upregulated 1alpha-25(OH) (2) D(3) mRNA followed by a timely delayed protein upregulation. Coincidently, enzymatic activity was enhanced significantly. The induction of the enzyme allowed for comparable differentiating effects of both vitamin D metabolites. CONCLUSION: Our experimental data provide a further mechanism for the involvement of the vitamin D signaling pathway in colonic epithelial cell differentiation by butyrate. The enhancement of 1alpha-25(OH) (2) D(3) followed by antiproliferative effects of the vitamin D prohormone in the Caco-2 cell line suggest that 25(OH) (2) D(3) in combination with butyrate may offer a new therapeutic approach for the treatment of colon cancer.