Baylor Scott and White Research Institute

Brown, M. N., D. C. Walters, M. A. Schmidt, J. Hill, A. McConnell, E. E. W. Jansen, G. S. Salomons, E. Arning, T. Bottiglieri, K. M. Gibson and J. B. Roullet (2019). “Maternal glutamine supplementation in murine succinic semialdehyde dehydrogenase deficiency, a disorder of gamma-aminobutyric acid metabolism.” J Inherit Metab Dis 42(5): 1030-1039.

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Murine succinic semialdehyde dehydrogenase deficiency (SSADHD) manifests with high concentrations of gamma-aminobutyric acid (GABA) and gamma-hydroxybutyrate (GHB) and low glutamine in the brain. To understand the pathogenic contribution of central glutamine deficiency, we exposed aldh5a1(-/-) (SSADHD) mice and their genetic controls (aldh5a1(+/+) ) to either a 4% (w/w) glutamine-containing diet or a glutamine-free diet from conception until postnatal day 30. Endpoints included brain, liver and blood amino acids, brain GHB, ataxia scores, and open field testing. Glutamine supplementation did not improve aldh5a1(-/-) brain glutamine deficiency nor brain GABA and GHB. It decreased brain glutamate but did not change the ratio of excitatory (glutamate) to inhibitory (GABA) neurotransmitters. In contrast, glutamine supplementation significantly increased brain arginine (30% for aldh5a1(+/+) and 18% for aldh5a1(-/-) mice), and leucine (12% and 18%). Glutamine deficiency was confirmed in the liver. The test diet increased hepatic glutamate in both genotypes, decreased glutamine in aldh5a1(+/+) but not in aldh5a1(-/-) , but had no effect on GABA. Dried bloodspot analyses showed significantly elevated GABA in mutants (approximately 800% above controls) and decreased glutamate (approximately 25%), but no glutamine difference with controls. Glutamine supplementation did not impact blood GABA but significantly increased glutamine and glutamate in both genotypes indicating systemic exposure to dietary glutamine. Ataxia and pronounced hyperactivity were observed in aldh5a1(-/-) mice but remained unchanged by the diet intervention. The study suggests that glutamine supplementation improves peripheral but not central glutamine deficiency in experimental SSADHD. Future studies are needed to fully understand the pathogenic role of brain glutamine deficiency in SSADHD.

Saravanan, P. B., S. Vasu, G. Yoshimatsu, C. M. Darden, X. Wang, J. Gu, M. C. Lawrence and B. Naziruddin (2019). “Differential expression and release of exosomal miRNAs by human islets under inflammatory and hypoxic stress.” Diabetologia Aug 1. [Epub ahead of print].

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AIMS/HYPOTHESIS: Pancreatic islets produce non-coding microRNAs (miRNAs) that regulate islet cell function and survival. Our earlier investigations revealed that human islets undergo significant damage due to various types of stresses following transplantation and release miRNAs. Here, we sought to identify and validate exosomal miRNAs (exo-miRNAs) produced by human islets under conditions of cellular stress, preceding loss of cell function and death. We also aimed to identify islet stress signalling pathways targeted by exo-miRNAs to elucidate potential regulatory roles in islet cell stress. METHODS: Human islets were subjected to proinflammatory cytokine and hypoxic cell stress and miRNA from exosomes was isolated for RNA sequencing and analysis. Stress-induced exo-miRNAs were evaluated for kinetics of expression and release by intact islets for up to 48 h exposure to cytokines and hypoxia. A subset of stress-induced exo-miRNAs were assessed for recovery and detection as biomarkers of islet cell stress in a diabetic nude mouse xenotransplant model and in patients undergoing total pancreatectomy with islet auto-transplantation (TPIAT). Genes and signalling pathways targeted by stress-induced exo-miRNAs were identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and direct interactions of miRNAs with downstream signalling targets were validated in human islet cells using the miRNA Tests for Read Analysis and Prediction (MirTrap) system. RESULTS: Global exo-miRNA sequencing revealed that 879 miRNA species were released from human islets and 190 islet exo-miRNAs were differentially expressed in response to proinflammatory cytokines, hypoxia or both. Release of exo-miRNAs hsa-miR-29b-3p and hsa-miR-216a-5p was detected within 6 h of exposure to cytokines and hypoxia. The remaining subset of stress-induced exo-miRNAs, including hsa-miR-148a-3p and islet cell damage marker hsa-miR-375, showed delayed release at 24-48 h, correlating with apoptosis and cell death. Stress and damage exo-miRNAs were significantly elevated in the circulation in human-to-mouse xenotransplant models and in human transplant recipients. Elevated blood exo-miRNAs negatively correlated with post-transplant islet function based on comparisons of stress and damage exo-miRNA indices with Secretory Unit of Islet Transplant Objects (SUITO) indices. KEGG analysis and further validation of exo-miRNA targets by MirTrap analysis revealed significant enrichment of islet mRNAs involved in phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase signalling pathways. CONCLUSIONS/INTERPRETATION: The study identifies exo-miRNAs differentially expressed and released by islets in response to damage and stress. These exo-miRNAs could serve as potential biomarkers for assessing islet damage and predicting outcomes in islet transplantation. Notably, exo-miRNAs 29b-3p and 216a-5p could be detected in islets prior to damage-released miRNAs and indicators of cellular apoptosis and death. Thus, these stress-induced exo-miRNAs may have potential diagnostic value for detecting early islet stress prior to progressive loss of islet cell mass and function. Further investigations are warranted to investigate the utility of these exo-miRNAs as early indicators of islet cell stress during prediabetic conditions.

Abu Ahmad, N., M. Raizman, N. Weizmann, B. Wasek, E. Arning, T. Bottiglieri, O. Tirosh and A. M. Troen (2019). “Betaine attenuates pathology by stimulating lipid oxidation in liver and regulating phospholipid metabolism in brain of methionine-choline-deficient rats.” FASEB 33(8): 9334-9349.

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Methyl-donor deficiency is a risk factor for neurodegenerative diseases. Dietary deficiency of the methyl-donors methionine and choline [methionine-choline-deficient (MCD) diet] is a well-established model of nonalcoholic steatohepatitis (NASH), yet brain metabolism has not been studied in this model. We hypothesized that supplemental betaine would protect both the liver and brain in this model and that any benefit to the brain would be due to improved liver metabolism because betaine is a methyl-donor in liver methylation but is not metabolically active in the brain. We fed male Sprague-Dawley rats a control diet, MCD diet, or betaine-supplemented MCD (MCD+B) diet for 8 wk and collected blood and tissue. As expected, betaine prevented MCD diet-induced NASH. However, contrary to our prediction, it did not appear to do so by stimulating methylation; the MCD+B diet worsened hyperhomocysteinemia and depressed liver methylation potential 8-fold compared with the MCD diet. Instead, it significantly increased the expression of genes involved in beta-oxidation: fibroblast growth factor 21 and peroxisome proliferator-activated receptor alpha. In contrast to that of the liver, brain methylation potential was unaffected by diet. Nevertheless, several phospholipid (PL) subclasses involved in stabilizing brain membranes were decreased by the MCD diet, and these improved modestly with betaine. The protective effect of betaine is likely due to the stimulation of beta-oxidation in liver and the effects on PL metabolism in brain.-Abu Ahmad, N., Raizman, M., Weizmann, N., Wasek, B., Arning, E., Bottiglieri, T., Tirosh, O., Troen, A. M. Betaine attenuates pathology by stimulating lipid oxidation in liver and regulating phospholipid metabolism in brain of methionine-choline-deficient rats.

Pannia, E., R. Hammoud, R. Kubant, J. Sa, N. Yang, M. Ho, D. Chatterjee, Z. Pausova, E. Arning, T. Bottiglieri and G. H. Anderson (2019). “5-Methyltetrahydrofolate in Maternal Diets Alters DNA Methylation Potential and Increases Later Life Weight Gain and Food Intake in Wistar Rat Dams and Female Offspring (P11-022-19).” Curr Dev Nutr Volume 3, Issue Supplement 1, June 2019. [Epub ahead of print].

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Objectives: Diet during pregnancy programs the mother and offspring post-weaning (PW). Folic acid (FA, synthetic folate) mediates DNA methylation (DNAm) reactions and high intakes, simulating those consumed by American women, lead to epigenetic dysregulation of energy metabolic pathways. 5-methyltetrahydrofolate (5MTHF), the bioactive folate form, has gained popularity as a supplement due to direct cellular uptake and utilization and does not increase unmetabolized FA (UMFA). However, a comparison of folate forms on in utero programming of offspring or maternal health has not been reported. Our objectives were to compare the effects of folate dose (low vs high) and form (FA vs 5MTHF) during pregnancy on DNAm potential, and the early and later PW phenotype of Wistar rat mothers and female offspring (mothers-to-be). Methods: Pregnant Wistar rats (n = 22/group) were fed an AIN93G diet with recommended FA (1X, 2mg/kg diet), 5X-FA or equimolar 5MTHF. Dams were fed 1X-FA during lactation and then dams and female pups were fed a high fat diet for 19 weeks. Weight gain (WG), food intake (FI), energy expenditure (EE), insulin resistance (IR), plasma 5MTHF, UMFA, homocysteine (tHcy), and hepatic S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and DNA methyltransferases (DNMT) activity at birth and PW were measured. Results: Dams fed 5MTHF diets had lower DNMT activity at birth and female pups had higher SAM: SAH ratios (P < 0.05) indicative of altered DNAm potential compared to FA diets. Plasma 5MTHF at birth was dose dependent with 5X diets leading to higher levels than 1X diets (P < 0.001) in dams and pups. In contrast, UMFA was only higher in 5X-FA dams. 5MTHF dams had higher tHcy at birth and were more IR at 1 week PW than FA fed dams (P < 0.05). In both dams and offspring, high 5MTHF also led to higher WG (> 15%, P < 0.001) and FI (> 5%, P < 0.001) compared to high FA diets up to 19 weeks. EE (P < 0.05) was higher suggesting a compensatory response to WG. 5X-MTHF dams, but not offspring, also had greater hepatic lipids (P < 0.05) than other groups. Conclusions: Folate dose and form during pregnancy affects DNAm potential at birth and early and later phenotype of dams and female offspring. High 5MTHF increases WG, FI and hepatic lipids PW suggesting it may not be the preferred form for prenatal supplements or additions to the food supply. Funding Sources: Supported by CIHR-INMD; EP supported by NSERC-CGS D.

Abu Ahmad, N., M. Raizman, N. Weizmann, B. Wasek, E. Arning, T. Bottiglieri, O. Tirosh and A. M. Troen (2019). “Betaine attenuates pathology by stimulating lipid oxidation in liver and regulating phospholipid metabolism in brain of methionine-choline-deficient rats.” FASEB Journal May 23. [Epub ahead of print].

Full text of this article.

Methyl-donor deficiency is a risk factor for neurodegenerative diseases. Dietary deficiency of the methyl-donors methionine and choline [methionine-choline-deficient (MCD) diet] is a well-established model of nonalcoholic steatohepatitis (NASH), yet brain metabolism has not been studied in this model. We hypothesized that supplemental betaine would protect both the liver and brain in this model and that any benefit to the brain would be due to improved liver metabolism because betaine is a methyl-donor in liver methylation but is not metabolically active in the brain. We fed male Sprague-Dawley rats a control diet, MCD diet, or betaine-supplemented MCD (MCD+B) diet for 8 wk and collected blood and tissue. As expected, betaine prevented MCD diet-induced NASH. However, contrary to our prediction, it did not appear to do so by stimulating methylation; the MCD+B diet worsened hyperhomocysteinemia and depressed liver methylation potential 8-fold compared with the MCD diet. Instead, it significantly increased the expression of genes involved in beta-oxidation: fibroblast growth factor 21 and peroxisome proliferator-activated receptor alpha. In contrast to that of the liver, brain methylation potential was unaffected by diet. Nevertheless, several phospholipid (PL) subclasses involved in stabilizing brain membranes were decreased by the MCD diet, and these improved modestly with betaine. The protective effect of betaine is likely due to the stimulation of beta-oxidation in liver and the effects on PL metabolism in brain.-Abu Ahmad, N., Raizman, M., Weizmann, N., Wasek, B., Arning, E., Bottiglieri, T., Tirosh, O., Troen, A. M. Betaine attenuates pathology by stimulating lipid oxidation in liver and regulating phospholipid metabolism in brain of methionine-choline-deficient rats.