Increased Abundance of Lactobacillales in the Colon of Beta-Adrenergic Receptor Knock Out Mouse Is Associated With Increased Gut Bacterial Production of Short Chain Fatty Acids and Reduced IL17 Expression in Circulating CD4+ Immune Cells

Emerging evidence suggests an associative link between gut dysbiosis, the autonomic nervous system (ANS) and the immune system in pathophysiology of neurogenic hypertension (HTN). However, the close interplay between these three systems presents us with difficulties in deciphering the cause-effect relationship in disease. The present study utilized beta 1 and 2 adrenergic receptor knock out (AdrB1tm1BkkAdrB2tm1Bkk/J KO) mice to isolate the effects of reduced overall sympathetic drive on gut microbiota and systemic immune system. We observed the following: (i) Diminished beta adrenergic signaling mainly reflects in shifts in the Firmicutes phyla, with a significant increase in abundance of largely beneficial Bacilli Lactobacillales in the KO mice; (ii) This was associated with increased colonic production of beneficial short chain fatty acids (SCFAs) butyrate, acetate and propionate, confirming functional microbiota shifts in the KO mice; (iii) Dampened systemic immune responses in the KO mice reflected in reduction on circulating CD4+.IL17+ T cells and increase in young neutrophils, both previously associated with shifts in the gut microbiota. Taken together, these observations demonstrate that reduced expression of beta adrenergic receptors may lead to beneficial shifts in the gut microbiota and dampened systemic immune responses. Considering the role of both in hypertension, this suggests that dietary intervention may be a viable option for manipulation of blood pressure via correcting gut dysbiosis

Structure and function of bacterial ion channels

KirBac channels are prokaryotic homologs of eukaryotic inwardly-rectifying potassium channels, which have served as models for gaining insight into the structure of eukaryotic channels. This thesis focuses on the structure-function relationship in these channels. The first part of this study concerns a novel KirBac channel, KirBac9.2, which contains a unique amino acid sequence in the place of the canonical GYG selectivity filter. Although expressed and purified in a stable and functional form, the protein did not form well-diffracting crystals. Functional studies suggest that KirBac9.2 is non-selective for monovalent cations and a random mutagenesis screen identified a number of activatory mutants in the cytoplasmic domains of the channel. A full electrophysiological investigation of KirBac9.2 channel function is beyond the scope of this study. However, initial studies suggest that it is possible to record currents from KirBac9.2 channels reconstituted into lipid bilayers. The second part of this thesis investigates KirBac3.1, which is a classical KirBac channel containing the consensus GYG sequence for potassium selectivity. Five high resolution structures of a mutant channel are reported, which suggest a new feature in the gating mechanism of KirBac3.1 where a rotation of the cytoplasmic domains is linked to a change in the electrostatic environment of the cytoplasmic cavity. In addition, a functional study of the KirBac3.1 showed that the channel is highly pH sensitive.</p

Electrical inhibition of identified anorexigenic POMC neurons by orexin/hypocretin.

Proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus (ARC) suppress appetite, and lack of POMC-derived peptides or electrical silencing of POMC neurons causes obesity. ARC POMC neurons are surrounded by nerve terminals containing the wakefulness-promoting peptides orexins/hypocretins, but whether orexin affects their electrical activity has not been tested directly. Here we identify living ARC POMC cells in mouse brain slices by targeted expression of green fluorescent protein. Using whole-cell patch-clamp recordings, we show that orexin suppresses the spontaneous action potential firing of these neurons. Orexin-induced inhibition involves membrane hyperpolarization, a decreased excitatory synaptic drive, and an increased frequency of GABAergic inputs. Our results suggest a reduction in the electrical activity of ARC POMC neurons, which is mediated by changes in presynaptic inputs, contributes to the appetite-enhancing action of orexins

Structure of a KirBac potassium channel with an open bundle crossing indicates a mechanism of channel gating

KirBac channels are prokaryotic homologs of mammalian inwardly rectifying (Kir) potassium channels, and recent crystal structures of both Kir and KirBac channels have provided major insight into their unique structural architecture. However, all of the available structures are closed at the helix bundle crossing, and therefore the structural mechanisms that control opening of their primary activation gate remain unknown. In this study, we engineered the inner pore-lining helix (TM2) of KirBac3.1 to trap the bundle crossing in an apparently open conformation and determined the crystal structure of this mutant channel to 3.05 Å resolution. Contrary to previous speculation, this new structure suggests a mechanistic model in which rotational 'twist' of the cytoplasmic domain is coupled to opening of the bundle-crossing gate through a network of inter-and intrasubunit interactions that involve the TM2 C-linker, slide helix, G-loop and the CD loop. © 2012 Nature America, Inc. All rights reserved

Interaction between Butyrate and Tumor Necrosis Factor α in Primary Rat Colonocytes

Butyrate, a short-chain fatty acid, is utilized by the gut epithelium as energy and it improves the gut epithelial barrier. More recently, it has been associated with beneficial effects on immune and cardiovascular homeostasis. Conversely, tumor necrosis factor alpha (TNFα) is a pro-inflammatory and pro-hypertensive cytokine. While butyrate and TNFα are both linked with hypertension, studies have not yet addressed their interaction in the colon. Here, we investigated the capacity of butyrate to modulate a host of effects of TNFα in primary rodent colonic cells in vitro. We measured ATP levels, cell viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), mitochondrial oxidative phosphorylation, and glycolytic activity in colonocytes following exposure to either butyrate or TNFα, or both. To address the potential mechanisms, transcripts related to oxidative stress, cell fate, and cell metabolism (Pdk1, Pdk2, Pdk4, Spr, Slc16a1, Slc16a3, Ppargc1a, Cs, Lgr5, Casp3, Tnfr2, Bax, Bcl2, Sod1, Sod2, and Cat) were measured, and untargeted liquid chromatography–tandem mass spectrometry (LC-MS/MS) was employed to profile the metabolic responses of colonocytes following exposure to butyrate and TNFα. We found that both butyrate and TNFα lowered cellular ATP levels towards a quiescent cell energy phenotype, characterized by decreased oxygen consumption and extracellular acidification. Co-treatment with butyrate ameliorated TNFα-induced cytotoxicity and the reduction in cell viability. Butyrate also opposed the TNFα-mediated decrease in MMP and mitochondrial-to-intracellular calcium ratios, suggesting that butyrate may protect colonocytes against TNFα-induced cytotoxicity by decreasing mitochondrial calcium flux. The relative expression levels of pyruvate dehydrogenase kinase 4 (Pdk4) were increased via co-treatment of butyrate and TNFα, suggesting the synergistic inhibition of glycolysis. TNFα alone reduced the expression of monocarboxylate transporters slc16a1 and slc16a3, suggesting effects of TNFα on butyrate uptake into colonocytes. Of the 185 metabolites that were detected with LC-MS, the TNFα-induced increase in biopterin produced the only significant change, suggesting an alteration in mitochondrial biogenesis in colonocytes. Considering the reports of elevated colonic TNFα and reduced butyrate metabolism in many conditions, including in hypertension, the present work sheds light on cellular interactions between TNFα and butyrate in colonocytes that may be important in understanding conditions of the colon

Sulfonylurea improves CNS function in a case of intermediate DEND syndrome caused by a mutation in KCNJ11.

BACKGROUND: A 12-week-old female presented with neonatal diabetes. Insulin therapy alleviated the diabetes, but the patient showed marked motor and mental developmental delay. The patient underwent genetic evaluation at the age of 6 years, prompted by reports that mutations in the KCNJ11 gene caused neonatal diabetes. INVESTIGATIONS: Genomic sequencing of the ATP-sensitive potassium (K(ATP)) channel gene KCNJ11 and in vitro functional analysis of the channel defect, and single-photon emission CT imaging before and after glibenclamide therapy. DIAGNOSIS: Genetic evaluation revealed a missense mutation (His46Leu) in KCNJ11, which encodes the Kir6.2 subunit of the K(ATP) channel, conferring reduced ATP sensitivity. Functional studies demonstrated that the mutant channels were strongly inhibited by the sulfonylurea tolbutamide. MANAGEMENT: Sulfonylurea (glibenclamide) treatment led to both improved glucose homeostasis and an increase in mental and motor function

Coincidence of a novel KCNJ11 missense variant R365H with a paternally inherited 6q24 duplication in a patient with transient neonatal diabetes.

OBJECTIVE: Neonatal diabetes is a heterogeneous group of disorders with diabetes manifestation in the first 6 months of life. The most common etiology in permanent neonatal diabetes is mutations of the ATP-sensitive K(+) channel subunits; in transient neonatal diabetes, chromosome 6q24 abnormalities are the most common cause. RESEARCH DESIGN AND METHODS: We report a sporadic case of diabetes without ketoacidosis diagnosed on the fourth day of life. RESULTS: Analysis of the KCNJ11 gene found a novel R365H mutation in the proband and her unaffected father. The functional analysis did not support pathogenicity of this variant. When the patient's diabetes remitted in the seventh month of life, the 6q24 region was analyzed and a paternally inherited duplication was identified. CONCLUSIONS: Our case reports a coincidental novel KCNJ11 variant in a patient with transient neonatal diabetes due to a 6q24 duplication, illustrating the difficulty in testing neonates before the clinical course of neonatal diabetes is known