Microbial composition, functionality, and stress resilience or susceptibility: unraveling sex-specific patterns.

BACKGROUND: Following exposure to traumatic stress, women are twice as likely as men to develop mood disorders. Yet, individual responses to such stress vary, with some people developing stress-induced psychopathologies while others exhibit resilience. The factors influencing sex-related disparities in affective disorders as well as variations in resilience remain unclear; however, emerging evidence suggests differences in the gut microbiota play a role. In this study, using the single prolonged stress (SPS) model of post-traumatic stress disorder, we investigated pre- and post-existing differences in microbial composition, functionality, and metabolites that affect stress susceptibility or resilience in each sex. METHODS: Male and female Sprague-Dawley rats were randomly assigned to control or SPS groups. Two weeks following SPS, the animals were exposed to a battery of behavioral tests and decapitated a day later. Based on their anxiety index, they were further categorized as SPS-resilient (SPS-R) or SPS-susceptible (SPS-S). On the day of dissection, cecum, and selected brain tissues were isolated. Stool samples were collected before and after SPS, whereas urine samples were taken before and 30 min into the SPS. RESULTS: Before SPS exposure, the sympathoadrenal axis exhibited alterations within male subgroups only. Expression of tight junction protein claudin-5 was lower in brain of SPS-S males, but higher in SPS-R females following SPS. Across the study, alpha diversity remained consistently lower in males compared to females. Beta diversity revealed distinct separations between male and female susceptible groups before SPS, with this separation becoming evident in the resilient groups following SPS. At the genus level, Lactobacillus, Lachnospiraceae_Incertae_Sedis, and Barnesiella exhibited sex-specific alterations, displaying opposing abundances in each sex. Additionally, sex-specific changes were observed in microbial predictive functionality and targeted functional modules both before and after SPS. Alterations in the microbial short-chain fatty acids (SCFAs), were also observed, with major and minor SCFAs being lower in SPS-susceptible males whereas branched-chain SCFAs being higher in SPS-susceptible females. CONCLUSION: This study highlights distinct pre- and post-trauma differences in microbial composition, functionality, and metabolites, associated with stress resilience in male and female rats. The findings underscore the importance of developing sex-specific therapeutic strategies to effectively address stress-related disorders. Highlights SPS model induces divergent anxiety and social behavioral responses to traumatic stress in both male and female rodents. SPS-resilient females displayed less anxiety-like behavior and initiated more interactions towards a juvenile rat than SPS-resilient males. Sex-specific pre-existing and SPS-induced differences in the gut microbial composition and predictive functionality were observed in susceptible and resilient rats. SPS-resilient males displayed elevated cecal acetate levels, whereas SPS-susceptible females exhibited heightened branched-chain SCFAs.

After experiencing traumatic stress, women are more likely than men to develop mood disorders like anxiety and depression. However, people's responses to trauma vary­some develop mental health issues while others remain resilient. Recent research suggests that the bacteria in the gut might play a role in these differences. In this study, using a rat model of post-traumatic stress disorder (PTSD), we investigated whether there are differences in gut bacteria between male and female rats before and after stress exposure. The study involved two groups of rats­one not exposed to stress (control) and the other exposed to a traumatic event (stressed). The rats' behavior was evaluated using different tests to determine who among the males and females were vulnerable to stress and who were resilient. We found that even before the stress, there were differences in the types of bacteria and their functions in the guts of male and female rats. These differences were also linked to how they responded to stress. Interestingly, the bacteria that were more abundant in resilient males were found to be more abundant in vulnerable females. Additionally, the traumatic stress affected these bacteria and the substances they produce differently in males and females. In essence, our study demonstrates that the types of gut bacteria, their functions, and their products contribute to stress resilience in different ways for male and female rats. This insight suggests that tailored treatments specifically targeting these differences could be specially effective in treating stress-related issues.

Effect of acetate supplementation on traumatic stress-induced behavioral impairments in male rats.

Gut microbiota and their metabolites have emerged as key players in the pathogenesis of neuropsychiatric disorders. Recently, we demonstrated that animals susceptible to Single Prolonged Stress (SPS) have an overall pro-inflammatory gut microbiota and significantly lower cecal acetate levels than SPS-resilient rats, which correlated inversely with the anxiety index. Here, we investigated whether the microbial metabolite, acetate, could ameliorate SPS-triggered impairments. Male rats were randomly divided into unstressed controls or groups exposed to SPS. The groups received continued oral supplementation of either 150 mM of sodium acetate or 150 mM of sodium chloride-matched water. Two weeks after SPS, a battery of behavioral tests was performed, and the animals were euthanized the following day. While not affecting the unstressed controls, acetate supplementation reduced the impact of SPS on body weight gain and ameliorated SPS-induced anxiety-like behavior and the impairments in social interaction, but not depressive-like behavior. These changes were accompanied by several beneficial effects of acetate supplementation. Acetate alleviated the stress response by reducing urinary epinephrine levels, induced epigenetic modification by decreasing histone deacetylase (HDAC2) gene expression, inhibited neuroinflammation by reducing the density of Iba1+ cells and the gene expression of IL-1ß in the hippocampus, and increased serum ß-hydroxybutyrate levels. The findings reveal a causal relationship between oral acetate treatment and mitigation of several SPS-induced behavioral impairments. Mechanistically, it impacted neuronal and metabolic pathways including changes in stress response, epigenetic modifications, neuroinflammation and showed novel link to ketone body production. The study demonstrates the preventive-therapeutic potential of acetate supplementation to alleviate adverse responses to traumatic stress.

Differences in gut microbiota associated with stress resilience and susceptibility to single prolonged stress in female rodents.

Exposure to traumatic stress is a major risk factor for the development of neuropsychiatric disorders in a subpopulation of individuals, whereas others remain resilient. The determinants of resilience and susceptibility remain unclear. Here, we aimed to characterize the microbial, immunological, and molecular differences between stress-susceptible and stress-resilient female rats before and after exposure to a traumatic experience. Animals were randomly divided into unstressed controls (n = 10) and experimental groups (n = 16) exposed to Single Prolonged Stress (SPS), an animal model of PTSD. Fourteen days later, all rats underwent a battery of behavioral tests and were sacrificed the following day to collect different organs. Stool samples were collected before and after SPS. Behavioral analyses revealed divergent responses to SPS. The SPS treated animals were further subdivided into SPS-resilient (SPS-R) and SPS-susceptible (SPS-S) subgroups. Comparative analysis of fecal 16S sequencing before and after SPS exposure indicated significant differences in the gut microbial composition, functionality, and metabolites of the SPS-R and SPS-S subgroups. In line with the observed distinct behavioral phenotypes, the SPS-S subgroup displayed higher blood-brain barrier permeability and neuroinflammation relative to the SPS-R and/or controls. These results indicate, for the first time, pre-existing and trauma-induced differences in the gut microbial composition and functionality of female rats that relate to their ability to cope with traumatic stress. Further characterization of these factors will be crucial for understanding susceptibility and fostering resilience, especially in females, who are more likely than males to develop mood disorders.

Resilience or susceptibility to traumatic stress: Potential influence of the microbiome.

Exposure to traumatic stress is a major risk factor for development of neuropsychiatric disorders in a sub-population of individuals, while others remain resilient. The mechanisms and contributing factors differentiating between these phenotypes are still unclear. We hypothesize that inter-individual differences in the microbial composition and function contribute to host resilience or susceptibility to stress-induced psychopathologies. The current study aimed to characterize gut microbial community before and after exposure to traumatic stress in an animal model of PTSD. Sprague-Dawley male rats were randomly divided into unstressed controls and experimental group subjected to Single Prolonged Stress (SPS). After 14 days, behavioral analyses were performed using Open Field, Social Interaction and Elevated Plus Maze tests. Based on the anxiety measures, the SPS group was further subdivided into resilient (SPS-R) and susceptible (SPS-S) cohorts. The animals were sacrificed after the last behavioral test and cecum, colon, hippocampus, and medial prefrontal cortex were dissected. Prior to SPS and immediately after Open Field test, fecal samples were collected from each rat for 16S V3-V4 ribosomal DNA sequencing, whereas urine samples were collected before SPS, 90 min into immobilization and on the day of sacrifice to measure epinephrine and norepinephrine levels. Analyses of the fecal microbiota revealed significant differences in microbial communities and in their predictive functionality among the groups before and after SPS stressors. Before SPS, the SPS-S subgroup harbored microbiota with an overall pro-inflammatory phenotype, whereas SPS-R subgroup had microbiota with an overall anti-inflammatory phenotype, with predictive functional pathways enriched in carbohydrate and lipid metabolism and decreased in amino acid metabolism and neurodegenerative diseases. After SPS, the gut microbial communities and their predictive functionality shifted especially in SPS cohorts, with volatility at the genus level correlating inversely with Anxiety Index. In line with the alterations seen in the gut microbiota, the levels of cecal short chain fatty acids were also altered, with SPS-S subgroup having significantly lower levels of acetate, valerate and caproate. The levels of acetate inversely correlated with Anxiety Index. Interestingly, urinary epinephrine and norepinephrine levels were also higher in the SPS-S subgroup at baseline and during stress, indicative of an altered sympathoadrenal stress axis. Finally, shorter colon (marker of intestinal inflammation) and a lower claudin-5 protein expression (marker for increased blood brain barrier permeability) were observed in the SPS-S subgroup. Taken together, our results suggest microbiota is a potential factor in predisposing subjects either to stress susceptibility or resilience. Moreover, SPS triggered significant shifts in the gut microbiota, their metabolites and brain permeability. These findings could lead to new therapeutic directions for PTSD possibly through the controlled manipulation of gut microbiota. It may enable early identification of individuals more likely to develop prolonged anxiogenic symptoms following traumatic stress.

Bacteria - derived short chain fatty acids restore sympathoadrenal responsiveness to hypoglycemia after antibiotic-induced gut microbiota depletion.

The microbiome co-evolved with their mammalian host over thousands of years. This commensal relationship serves a pivotal role in various metabolic, physiological, and immunological processes. Recently we discovered impaired adrenal catecholamine stress responses in germ-free mice suggesting developmental modification of the reflex arc or absence of an ongoing microbiome signal. To determine whether maturational arrest or an absent bacteria-derived metabolite was the cause, we tested whether depleting gut microbiome in young adult animals could also alter the peripheral stress responses to insulin-induced hypoglycemia. Groups of C57Bl6 male mice were given regular water (control) or a cocktail of non-absorbable broad-spectrum antibiotics (Abx) in the drinking water for two weeks before injection with insulin or saline. Abx mice displayed a profound decrease in microbial diversity and abundance of Bacteroidetes and Firmicutes, plus a markedly enlarged caecum and no detectable by-products of bacterial fermentation (sp. short chain fatty acids, SCFA). Tonic and stress-induced epinephrine levels were attenuated. Recolonization (Abx + R) restored bacterial diversity, but not the sympathoadrenal system responsiveness or caecal acetate, propionate and butyrate levels. In contrast, corticosterone (HPA) and glucagon (parasympathetic) resting values and responses to hypoglycemia remained similar across all conditions. Oral supplementation with SCFA improved epinephrine responses to hypoglycaemia. Whole genome shotgun sequence profiling of fecal samples from control, Abx and Abx + R cohorts identified nine microbes (SCFA producers) absent from both Abx and Abx + R groups. These results implicate gut microbiome depletion plus its attendant reduction in SCFA signalling in adversely affecting the release of epinephrine in response to hypoglycemia. We speculate that regardless of postnatal age, a mutable microbiome messaging system exists throughout life. Unravelling these mechanisms could lead to new therapeutic possibilities through controlled manipulation of the gut microbiota and its ability to alter systemic neurotransmitter responsiveness.

Absence of gut microbial colonization attenuates the sympathoadrenal response to hypoglycemic stress in mice: implications for human neonates.

BACKGROUND: Gut microbiota plays an important role during early development via bidirectional gut-brain signaling. Catecholamines provide a survival advantage allowing adaptation to common postnatal stressors. We aimed to explore the potential link between gut microbiota/gut-derived metabolites and sympathoadrenal stress responsivity. METHODS: The effect of insulin-induced hypoglycemia was compared in mice with (control, adapted control) and without microbiome (germ-free, GF). Counter-regulatory hormones were analyzed in urine and plasma. Adrenal gene expression levels were evaluated and correlated to cecal short chain fatty acids (SCFA) content. RESULTS: There was a significant association between absent microbiota/SCFA and epinephrine levels at baseline and after stress. Corticosterone (hypothalamic-pituitary-adrenal axis) and glucagon release (parasympathetic signaling) were similar in all groups. Hypoglycemia-induced c-Fos (marker of trans-synaptic neuronal activation) in both conditions. Delayed increases in adrenal tyrosine hydroxylase and neuropeptide Y messenger RNA were observed in GF mice. Transcriptome analysis provided insight into underlying mechanisms for attenuated epinephrine production and release. CONCLUSION: Lack of microbiome selectively impaired adrenal catecholamine responses to hypoglycemia. We speculate that absent/delayed acquisition of flora (e.g., after antibiotic exposure) may compromise sympathoadrenal stress responsivity. Conversely, controlled manipulation of the intestinal microflora may provide a novel therapeutic opportunity to improve survival and overall health in preterm neonates.

Correction: Whole genome expression profiling associates activation of unfolded protein response with impaired production and release of epinephrine after recurrent hypoglycemia.

[This corrects the article DOI: 10.1371/journal.pone.0172789.].

Whole genome expression profiling associates activation of unfolded protein response with impaired production and release of epinephrine after recurrent hypoglycemia.

Recurrent hypoglycemia can occur as a major complication of insulin replacement therapy, limiting the long-term health benefits of intense glycemic control in type 1 and advanced type 2 diabetic patients. It impairs the normal counter-regulatory hormonal and behavioral responses to glucose deprivation, a phenomenon known as hypoglycemia associated autonomic failure (HAAF). The molecular mechanisms leading to defective counter-regulation are not completely understood. We hypothesized that both neuronal (excessive cholinergic signaling between the splanchnic nerve fibers and the adrenal medulla) and humoral factors contribute to the impaired epinephrine production and release in HAAF. To gain further insight into the molecular mechanism(s) mediating the blunted epinephrine responses following recurrent hypoglycemia, we utilized a global gene expression profiling approach. We characterized the transcriptomes during recurrent (defective counter-regulation model) and acute hypoglycemia (normal counter-regulation group) in the adrenal medulla of normal Sprague-Dawley rats. Based on comparison analysis of differentially expressed genes, a set of unique genes that are activated only at specific time points after recurrent hypoglycemia were revealed. A complementary bioinformatics analysis of the functional category, pathway, and integrated network indicated activation of the unfolded protein response. Furthermore, at least three additional pathways/interaction networks altered in the adrenal medulla following recurrent hypoglycemia were identified, which may contribute to the impaired epinephrine secretion in HAAF: greatly increased neuropeptide signaling (proenkephalin, neuropeptide Y, galanin); altered ion homeostasis (Na+, K+, Ca2+) and downregulation of genes involved in Ca2+-dependent exocytosis of secretory vesicles. Given the pleiotropic effects of the unfolded protein response in different organs, involved in maintaining glucose homeostasis, these findings uncover broader general mechanisms that arise following recurrent hypoglycemia which may afford clinicians an opportunity to modulate the magnitude of HAAF syndrome.

Modulation of Immunological Pathways in Autistic and Neurotypical Lymphoblastoid Cell Lines by the Enteric Microbiome Metabolite Propionic Acid.

Propionic acid (PPA) is a ubiquitous short-chain fatty acid which is a fermentation product of the enteric microbiome and present or added to many foods. While PPA has beneficial effects, it is also associated with human disorders, including autism spectrum disorders (ASDs). We previously demonstrated that PPA modulates mitochondrial dysfunction differentially in subsets of lymphoblastoid cell lines (LCLs) derived from patients with ASD. Specifically, PPA significantly increases mitochondrial function in LCLs that have mitochondrial dysfunction at baseline [individuals with autistic disorder with atypical mitochondrial function (AD-A) LCLs] as compared to ASD LCLs with normal mitochondrial function [individuals with autistic disorder with normal mitochondrial function (AD-N) LCLs] and control (CNT) LCLs. PPA at 1 mM was found to have a minimal effect on expression of immune genes in CNT and AD-N LCLs. However, as hypothesized, Panther analysis demonstrated that 1 mM PPA exposure at 24 or 48 h resulted in significant activation of the immune system genes in AD-A LCLs. When the effect of PPA on ASD LCLs were compared to the CNT LCLs, both ASD groups demonstrated immune pathway activation, although the AD-A LCLs demonstrate a wider activation of immune genes. Ingenuity Pathway Analysis identified several immune-related pathways as key Canonical Pathways that were differentially regulated, specifically human leukocyte antigen expression and immunoglobulin production genes were upregulated. These data demonstrate that the enteric microbiome metabolite PPA can evoke atypical immune activation in LCLs with an underlying abnormal metabolic state. As PPA, as well as enteric bacteria which produce PPA, have been implicated in a wide variety of diseases which have components of immune dysfunction, including ASD, diabetes, obesity, and inflammatory diseases, insight into this metabolic modulator may have wide applications for both health and disease.

Posttranscriptional regulation of adrenal TH gene expression contributes to the maladaptive responses triggered by insulin-induced recurrent hypoglycemia.

Acute metabolic stress such as insulin-induced hypoglycemia triggers a counterregulatory response during which the release of catecholamines (epinephrine), the activation of tyrosine hydroxylase (TH) enzyme and subsequent compensatory catecholamine biosynthesis occur in the adrenal medulla. However, recurrent exposure to hypoglycemia (RH), a consequence of tight glycemic control in individuals with type 1 and type 2 diabetes compromises this physiological response. The molecular mechanisms underlying the maladaptive response to repeated glucose deprivation are incompletely understood. We hypothesize that impaired epinephrine release following RH reflects altered regulation of adrenal catecholamine biosynthesis. To test this hypothesis, we compared the effect of single daily (RH) and twice-daily episodes of insulin-induced hypoglycemia (2RH) on adrenal epinephrine release and production in normal rats. Control animals received saline injections under similar conditions (RS and 2RS, respectively). Following 3 days of treatment, we assessed the counterregulatory hormonal responses during a hypoglycemic clamp. Changes in adrenal TH gene expression were also analyzed. The counterregulatory responses, relative TH transcription and TH mRNA levels and Ser40-TH phosphorylation (marker for enzyme activation) were induced to a similar extent in RS, 2RS, and RH groups. In contrast, epinephrine and glucagon responses were attenuated in the 2RH group and this was associated with a limited elevation of adrenal TH mRNA, rapid inactivation of TH enzyme and no significant changes in TH protein. Our results suggest that novel posttranscriptional mechanisms controlling TH mRNA and activated TH enzyme turnover contribute to the impaired epinephrine responses and may provide new therapeutic targets to prevent HAAF.

Enteric bacterial metabolites propionic and butyric acid modulate gene expression, including CREB-dependent catecholaminergic neurotransmission, in PC12 cells--possible relevance to autism spectrum disorders.

Alterations in gut microbiome composition have an emerging role in health and disease including brain function and behavior. Short chain fatty acids (SCFA) like propionic (PPA), and butyric acid (BA), which are present in diet and are fermentation products of many gastrointestinal bacteria, are showing increasing importance in host health, but also may be environmental contributors in neurodevelopmental disorders including autism spectrum disorders (ASD). Further to this we have shown SCFA administration to rodents over a variety of routes (intracerebroventricular, subcutaneous, intraperitoneal) or developmental time periods can elicit behavioral, electrophysiological, neuropathological and biochemical effects consistent with findings in ASD patients. SCFA are capable of altering host gene expression, partly due to their histone deacetylase inhibitor activity. We have previously shown BA can regulate tyrosine hydroxylase (TH) mRNA levels in a PC12 cell model. Since monoamine concentration is known to be elevated in the brain and blood of ASD patients and in many ASD animal models, we hypothesized that SCFA may directly influence brain monoaminergic pathways. When PC12 cells were transiently transfected with plasmids having a luciferase reporter gene under the control of the TH promoter, PPA was found to induce reporter gene activity over a wide concentration range. CREB transcription factor(s) was necessary for the transcriptional activation of TH gene by PPA. At lower concentrations PPA also caused accumulation of TH mRNA and protein, indicative of increased cell capacity to produce catecholamines. PPA and BA induced broad alterations in gene expression including neurotransmitter systems, neuronal cell adhesion molecules, inflammation, oxidative stress, lipid metabolism and mitochondrial function, all of which have been implicated in ASD. In conclusion, our data are consistent with a molecular mechanism through which gut related environmental signals such as increased levels of SCFA's can epigenetically modulate cell function further supporting their role as environmental contributors to ASD.

Partial blockade of nicotinic acetylcholine receptors improves the counterregulatory response to hypoglycemia in recurrently hypoglycemic rats.

Recurrent exposure to hypoglycemia can impair the normal counterregulatory hormonal responses that guard against hypoglycemia, leading to hypoglycemia unawareness. This pathological condition known as hypoglycemia-associated autonomic failure (HAAF) is the main adverse consequence that prevents individuals with type 1 diabetes mellitus from attaining the long-term health benefits of tight glycemic control. The underlying molecular mechanisms responsible for the progressive loss of the epinephrine response to subsequent bouts of hypoglycemia, a hallmark sign of HAAF, are largely unknown. Normally, hypoglycemia triggers both the release and biosynthesis of epinephrine through activation of nicotinic acetylcholine receptors (nAChR) on the adrenal glands. We hypothesize that excessive cholinergic stimulation may contribute to impaired counterregulation. Here, we tested whether administration of the nAChR partial agonist cytisine to reduce postganglionic synaptic activity can preserve the counterregulatory hormone responses in an animal model of HAAF. Compared with nicotine, cytisine has limited efficacy to activate nAChRs and stimulate epinephrine release and synthesis. We evaluated adrenal catecholamine production and secretion in nondiabetic rats subjected to two daily episodes of hypoglycemia for 3 days, followed by a hyperinsulinemic hypoglycemic clamp on day 4. Recurrent hypoglycemia decreased epinephrine responses, and this was associated with suppressed TH mRNA induction (a measure of adrenal catecholamine synthetic capacity). Treatment with cytisine improved glucagon responses as well as epinephrine release and production in recurrently hypoglycemic animals. These data suggest that pharmacological manipulation of ganglionic nAChRs may be promising as a translational adjunctive therapy to avoid HAAF in type 1 diabetes mellitus.

Valproic acid regulates catecholaminergic pathways by concentration-dependent threshold effects on TH mRNA synthesis and degradation.

The spectrum of neurological conditions and psychiatric disorders affected by valproic acid (VPA) ranges from control of seizure and mood disorders to migraine, neuropathic pain, and even congenital malformations and autism. While widely used clinically, the mechanism(s) of action of VPA is not completely understood. Emerging evidence indicates that brain noradrenergic systems contribute to the symptoms of mood disorders and may involve regulation of tyrosine hydroxylase (TH) expression, the rate-limiting enzyme in the biosynthesis of dopamine, norepinephrine and epinephrine. We previously showed that the structurally related short chain fatty acid sodium butyrate (SB) induces TH transcription and alters TH mRNA stability in PC12 cells. The present study was undertaken to determine whether the branched short chain fatty acid VPA could also regulate TH gene expression in vitro. Similar to SB, VPA induced TH transcription at all concentrations tested. VPA-stimulated transcription was significantly attenuated by introducing point mutations in either the canonical cAMP- or in the butyrate-response elements of the TH promoter; or by co-expression of dominant-negative forms of CREB. As with SB, increasing concentrations of VPA demonstrated opposing effects on TH mRNA and protein abundance: elevation of both at low (0.1 mM) but attenuation at concentrations higher than 0.5 mM. This concentration-dependence is consistent with a novel and previously unrecognized cellular/molecular drug regulatory step at the level of TH mRNA stability. Thus, the therapeutic efficacy of VPA might be related to its ability to regulate TH mRNA and protein levels, and thereby central catecholaminergic-dependent behavioral pathways.

Differential regulation of the tyrosine hydroxylase and enkephalin neuropeptide transmitter genes in rat PC12 cells by short chain fatty acids: concentration-dependent effects on transcription and RNA stability.

At physiologic concentrations, butyrate regulates the expression of individual genes involving at least three mechanisms: (i) through induction of cis- and trans-acting butyrate-dependent transcription factors for selected genes, (ii) by inhibition of histone deacetylation and attendant chromatin remodeling and (iii) by affecting turnover of mRNAs. Our previous work illustrated gradual accumulation of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis and the neuropeptide transmitter proenkephalin (ppEnk) in butyrate-differentiated PC12 cells (Nankova, B.B., Chua, J., Mishra, R., Kobasiuk, C.D., La Gamma, E.F. 2003. Nicotinic induction of preproenkephalin and tyrosine hydroxylase gene expression in butyrate-differentiated rat PC12 cells: a model for adaptation to gut-derived environmental signals. Pediatr. Res. 53, 113-118.). However, at higher physiological concentrations (6 mM), TH mRNA levels are significantly reduced while ppEnk mRNA transcripts remained elevated. These differential effects suggest suppression of endogenous TH gene transcription, targeted degradation of TH mRNA or both. By using nuclear run-on assays, we found that transcription increased for both endogenous TH and ppEnk genes, even at time points and concentrations when reduced steady-state levels of TH mRNA were observed. The reduction in TH mRNA was blocked by cycloheximide consistent with a protein-dependent mechanism. We also observed a dose-dependent accumulation of luciferase reporter molecules driven by TH promoter in transient transfection experiments, data that provide additional support for separate regulatory pathways. Significantly, butyrate-dependent decreases in TH mRNA were also reflected in a reduction in TH protein. Our results suggest a novel mode of regulation for TH by butyrate operating via both transcriptional and post-transcriptional mechanisms. We speculate that, depending on plasma concentrations of butyrate, this naturally occurring signaling molecule can function as an in vivo molecular switch to alter levels of TH mRNA, its protein and thus the biosynthesis of endogenous catecholamines.

Short chain fatty acids induce TH gene expression via ERK-dependent phosphorylation of CREB protein.

Butyrate modulates specific gene expression through various second-messenger signal transduction systems including activation of the PKA/cAMP pathway (Decastro, M., Nankova, B.B., Shah, P., Patel, P., Mally, P.V., Mishra, R., La Gamma, E.F., 2005. Short chain fatty acids regulate tyrosine hydroxylase gene expression through a cAMP-dependent signaling pathway, Brain Res. Mol. Brain Res. 142 28-38; Mally, P., Mishra, R., Gandhi, S., Decastro, M.H., Nankova, B.B., Lagamma, E.F., 2004. Stereospecific regulation of tyrosine hydroxylase and proenkephalin genes by short-chain fatty acids in rat PC12 cells, Pediatr. Res. 55 847-854). In the current report, we provide additional evidence that exposure to butyrate causes a rapid activation of the MAP kinase pathway, associated with increased phosphorylation of CREB. Under these conditions, no changes in relative amounts of CREB protein were observed by Western blot. Pre-treatment with the MAPK specific inhibitor (U0126) or the adenylate cyclase inhibitor dideoxyadenosine (ddA) abolished the butyrate-induced: (i) accumulation of TH mRNA, (ii) the phosphorylation of ERK1/2 as well as (iii) CREB phosphorylation. PC12 cells transfected with a TH promoter-luciferase reporter gene showed a robust induction in response to butyrate that was significantly reduced after co-transfection of either of two dominant-negative CREB expression vectors. Nuclear run-on assays demonstrated that butyrate increases endogenous TH gene transcription. We conclude that the initial steps of butyrate-induced gene activation are mediated through the CREB/CREB family of transcription factors which are coupled to both the MAP kinase and cAMP-dependent second messenger systems. Our data delineate a molecular mechanism through which short chain fatty acid's, their related drug-congeners (e.g., valproate) or even diet-derived butyrate (from fermentation of carbohydrates in the gut) can in principle, modulate brain catecholaminergic systems by modifying TH gene expression, dopaminergic levels and the corresponding animal behavior. These molecular relationships also offer a plausible explanation of how the well-recognized clinical effects of ketogenic diets can alter human behavior via the same central mechanisms.

Short chain fatty acids regulate tyrosine hydroxylase gene expression through a cAMP-dependent signaling pathway.

Multiple intracellular and extracellular regulatory factors affect transcription of the tyrosine hydroxylase (TH) gene encoding the rate-limiting enzyme in the biosynthesis of the neurotransmitters dopamine, norepinephrine and epinephrine. Short chain fatty acids like butyrate are known to alter TH gene expression, but the mechanism of action is unknown. In this report, transient transfection assays identified the proximal TH promoter to contain sufficient genetic information to confer butyrate responsiveness to a reporter gene. Deletion studies and gel shift analyses revealed that the promoter region spanning the cAMP response element is an absolute requirement for transcriptional activation by butyrate. The branched short chain fatty acid valproate is used for seizure control in humans. Significantly, it has a similar aliphatic structure to butyrate, and it was found to have similar effects on TH in PC12 cells. Site-directed mutagenesis indicated that the effects of both fatty acids were mediated through the canonical CRE. Butyrate treatment also resulted in CREB phosphorylation without changing CREB protein levels. The increased phosphorylation of CREB correlated with accumulation of TH mRNA. The adenylate cyclase inhibitor dideoxyadenosine blocked both CREB phosphorylation and accumulation of TH mRNA. The data are consistent with the conclusion that butyrate induces post-translational modifications of pre-existing CREB molecules in a cAMP/PKA-dependent manner to alter TH transcription. These results support the role of butyrate as a novel exogenous regulatory factor in TH gene expression. Our data delineate a molecular mechanism through which diet-derived environmental signals (e.g. butyrate) can modulate catecholaminergic systems by affecting TH gene transcription.

Butyrate, a gut-derived environmental signal, regulates tyrosine hydroxylase gene expression via a novel promoter element.

Butyrate is a diet-derived, gut fermentation product with an array of effects on cultured mammalian cells including inhibition of proliferation, induction of differentiation and regulation of gene expression. We showed that physiological concentrations of butyrate can regulate transcription of tyrosine hydroxylase (TH) and preproenkephalin (ppEnk) gene in PC12 cells. In promoter deletion studies, electrophoretic mobility shift assays and by site-directed mutagenesis, we identified a novel butyrate response element (BRE) in the 5' upstream region of the rat TH gene, homologous to the previously mapped motif in the ppEnk promoter. No such enhancers were found in DBH or PNMT promoters, and both catecholamine system-related gene promoters were unaffected by butyrate. The BRE motif interacts with nuclear proteins in a sequence-specific manner, shows binding potentiation in butyrate-differentiated PC12 cells and bound protein(s) are competed away with TH-CRE oligonucleotides or by the addition of CREB-specific antibodies, suggesting involvement of CREB or CREB-related transcription factors. Moreover, single point mutation in the distal BRE abolished binding of transcription factors and reduced the response to butyrate in transient transfection studies. The canonical CRE motif of the TH promoter was also found necessary for transcriptional activation of the TH gene by butyrate. Our data identified a novel functional element in the promoter of both the TH and ppEnk genes mediating transcriptional responses to butyrate. Dietary butyrate may have an extended role in the control of catecholamine and endogenous opioid production at the level of TH and ppEnk gene transcription neuronal plasticity, cardiovascular functions, stress adaptation and behavior.

Molecular regulation of gene expression of catecholamine biosynthetic enzymes by stress: sympathetic ganglia versus adrenal medulla.

Stress induces tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) gene expression in sympathetic ganglia and adrenal medulla (AM). However, distinct molecular mechanisms appear to regulate these genes in these locations. The elevation of TH mRNA in response to single immobilization stress (IMO) in AM is robust, but transient, while the induction of TH and DBH mRNAs in sympathetic ganglia is slower and more long lasting. Injections of adrenocorticotropic hormone (ACTH) elicited induction of TH and DBH gene expression in rat sympathetic ganglia, but not in AM. The superior cervical (SCG) and stellate (StG) ganglia, but not AM, were found to express mRNA for the MC-2 receptor, the major ACTH responsive receptor in adrenal cortex. IMO led to increase in MC-2 receptor mRNA levels in SCG. Thus, ACTH, via the MC-2 receptor, may be directly involved in the stress-elicited regulation of norepinephrine biosynthesis in sympathetic ganglia. The signaling pathways triggered by IMO differed in these locations. In AM, IMO triggered activation of the MAP kinase, JNK, and induction of AP1 factors, Egr1 and phosphorylation of CREB. In contrast in the SCG, with IMO we did not observe changes in JNK and little binding to the AP1 motif of the TH promoter. However, there was an increase in CREB binding to the CRE site of the TH promoter. The results reveal differential mechanisms of regulation of catecholamine biosynthetic enzymes by stress in two components of the sympathoadrenal system and should provide basis for possible selective pharmacologic interventions.

Role of Ca2+ in induction of neurotransmitter-related gene expression by butyrate.

We examined the effect of butyrate on neurotransmitter-related gene expression and calcium homeostasis in PC12 cells. Pretreatment with Ca2+ chelators (EGTA or BAPTA-AM) attenuated the butyrate-triggered accumulation of TH and ppEnk mRNA indicating that Ca2+ plays a role in butyrate-induced regulation of neuronal genes. Butyrate alone did not alter intracellular Ca2+ levels as determined by Fura-PE3 fluorescence; however, pretreatment with butyrate (18-24 h) reduced the first Ca2+ peak and prevented the second sustained rise in [Ca2+]i as induced by nicotine or ryanodine. In contrast, butyrate had no effect on Ca2+ transients when added shortly before or during nicotine or ryanodine stimulation. These results suggest that chronic butyrate exposure can modulate cell responses by affecting intracellular Ca2+ signaling.

Stereospecific regulation of tyrosine hydroxylase and proenkephalin genes by short-chain fatty acids in rat PC12 cells.

Circulating short-chain fatty acids (SCFAs) are primarily derived from bacterial fermentation of carbohydrates in the colon where they function as physiologic modulators of epithelial cell maturation. Butyrate has been shown to induce tyrosine hydroxylase, the rate-limiting enzyme of catecholamine synthesis, and enkephalin neuropeptide gene transcription, suggesting a role in perinatal sympathoadrenal stress-adaptation. We sought to determine whether there were SCFA structural requirements for this effect. Nine biologically relevant SCFAs and butyrate derivatives were tested in an in vitro model (PC12, rat pheochromocytoma cells) for their ability to regulate neurotransmitter-related gene expression. Our results revealed that among all the studied SCFAs, only propionate and butyrate increased tyrosine hydroxylase and proenkephalin mRNA levels. The functional activity was selective to the carbon atom chain length and associated with the presence of an ethyl moiety in the carbon atom backbone chain. Modifications or absence of this domain affected the gene induction response, suggesting a receptor-mediated mechanism(s). Moreover, propionate, butyrate, and the drug 4-phenylbutyrate were each shown to regulate transmitter genes via at least three independent mechanisms: histone hyperacetylation, cAMP signaling, or peroxisome proliferator-activated receptor gamma-mediated pathways. Thus, the biologic impact of SCFAs on catecholaminergic and opioid systems depend on the activation of SCFA-specific, dose-specific, and gene-specific molecular mechanisms. We speculate that 1) circulating levels of SCFAs may influence sympathoadrenal transmitter biosynthesis and hence whole animal stress-adaptive responsiveness after birth, and 2) the adverse effects of antibiotics on delayed acquisition of postnatal gut flora may affect this apparent evolutionary advantage of gut colonization.