High-fat diet-induced obesity alters nitric oxide-mediated neuromuscular transmission and smooth muscle excitability in the mouse distal colon.

We tested the hypothesis that colonic enteric neurotransmission and smooth muscle cell (SMC) function are altered in mice fed a high-fat diet (HFD). We used wild-type (WT) mice and mice lacking the ß1-subunit of the BK channel (BKß1 (-/-)). WT mice fed a HFD had increased myenteric plexus oxidative stress, a 28% decrease in nitrergic neurons, and a 20% decrease in basal nitric oxide (NO) levels. Circular muscle inhibitory junction potentials (IJPs) were reduced in HFD WT mice. The NO synthase inhibitor nitro-l-arginine (NLA) was less effective at inhibiting relaxations in HFD compared with control diet (CD) WT mice (11 vs. 37%, P < 0.05). SMCs from HFD WT mice had depolarized membrane potentials (-47 ± 2 mV) and continuous action potential firing compared with CD WT mice (-53 ± 2 mV, P < 0.05), which showed rhythmic firing. SMCs from HFD or CD fed BKß1 (-/-) mice fired action potentials continuously. NLA depolarized membrane potential and caused continuous firing only in SMCs from CD WT mice. Sodium nitroprusside (NO donor) hyperpolarized membrane potential and changed continuous to rhythmic action potential firing in SMCs from HFD WT and BKß1 (-/-) mice. Migrating motor complexes were disrupted in colons from BKß1 (-/-) mice and HFD WT mice. BK channel α-subunit protein and ß1-subunit mRNA expression were similar in CD and HFD WT mice. We conclude that HFD-induced obesity disrupts inhibitory neuromuscular transmission, SMC excitability, and colonic motility by promoting oxidative stress, loss of nitrergic neurons, and SMC BK channel dysfunction.

Sex-related differences in small intestinal transit and serotonin dynamics in high-fat-diet-induced obesity in mice.

Obesity alters gastrointestinal (GI) motility and 5-HT signalling. Altered 5-HT signalling disrupts control of GI motility. Levels of extracellular 5-HT depend on enterochromaffin (EC) cell release and serotonin transporter (SERT) uptake. We assessed GI transit and 5-HT signalling in the jejunum of normal and obese mice. Male and female mice were fed a control diet (CD; 10% of kilocalories as fat) or a high-fat diet (HFD; 60% of kilocalories as fat). Gastrointestinal transit was increased in male HFD-fed and female CD-fed compared with male CD-fed mice. The 5-HT3 receptor blocker, alosetron, increased gastric emptying in male CD-fed mice, but decreased transit in female CD-fed mice. The 5-HT-induced jejunal longitudinal muscle contractions in vitro were similar in all mice. In contrast to male CD-fed mice, 5-HT uptake (measured using continuous amperometry in vitro) in male HFD-fed mice was fluoxetine insensitive, yet sensitive to cocaine and the dopamine transporter (DAT) blocker GBR 12909. Immunoreactivity for DAT was present in the mucosa, and protein levels were greater in male HFD-fed compared with CD-fed mice. Extracellular 5-HT and mucosal 5-hydroxyindolacetic acid (5-HT metabolite) were similar in male HFD-fed compared with CD-fed mice. 5-Hydroxytryptamine uptake was fluoxetine sensitive in all females. Greater SERT protein, decreased extracellular 5-HT and greater mucosal 5-hydroxyindolacetic acid were observed in female HFD-fed compared with CD-fed mice. Mucosal 5-HT and EC cell numbers were similar in CD-fed and HFD-fed mice of both sexes; female 5-HT and EC cell numbers were increased compared with males. The HFD did not alter plasma sex hormone levels in any mice. Overall, obesity alters GI transit and 5-HT signalling in a sex-dependent manner.

Noninvasive biomarkers of necrotizing enterocolitis.

Necrotizing enterocolitis is an acute inflammatory disease, which primarily affects preterm infants, and is a leading cause of morbidity and mortality in the neonatal intensive care unit. Unfortunately, necrotizing enterocolitis can be difficult to distinguish from other diseases and clinical conditions especially during the early course of the disease. This diagnostic uncertainty is particularly relevant to clinical evaluation and medical management and potentially leads to unnecessary and extended periods of cessation of enteral feedings and prolonged courses of parenteral nutrition and antibiotics. Biomarkers are molecular indicators of a disease process, diagnosis, prognosis and can be used to monitor the effects of disease management. Historically, there has been a paucity of reliable and robust biomarkers for necrotizing enterocolitis. However, several studies have recently identified promising biomarkers. Noninvasive samples for biomarker measurement are preferred and may have certain advantages in the preterm infant. In this review article, we focus on recent exciting and promising discoveries in noninvasive biomarkers for necrotizing enterocolitis.

Necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is an acute inflammatory disease of the intestine which primarily affects preterm infants and is a leading cause of morbidity and mortality in the neonatal intensive care unit. From a clinical standpoint, and during the early course of the disease, NEC can be difficult to distinguish from other diseases and conditions common to the preterm infant, and this warrants the need for specific disease biomarkers. The pathogenesis of NEC is only partly understood but likely involves an altered intestinal barrier immune response to feeding and the developing microbiome. Recent evidence points toward a role of the enteric nervous system in NEC pathogenesis. In this issue, Meister and colleagues use a rodent model of NEC to demonstrate that NEC is associated with diminished vagal tone, as determined by decreased high-frequency heart rate variability (HF-HRV), and altered myenteric nitrergic inhibitory neurotransmission. These results augment their previous findings that describe decreased HF-HRV in human preterm infants with NEC. This mini-review provides a brief summary of clinical and pathophysiologic aspects of NEC with focus on certain aspects of neurogastroenterology.

Nonimmune hydrops fetalis and hepatitis in a neonate with congenital human immunodeficiency virus infection.

We present a case report of a term neonate with congenital human immunodeficiency virus (HIV) infection born with nonimmune hydrops fetalis who developed hepatitis shortly after birth. Maternal HIV infection was diagnosed after delivery. An extensive evaluation for known causes of nonimmune hydrops, both infectious and noninfectious, was negative. After beginning highly active antiretroviral therapy, hepatitis resolved and the HIV viral load became undetectable. We believe this is the first report of nonimmune hydrops fetalis and hepatitis in an infant with congenital HIV infection.

Western blot analysis of BK channel ß1-subunit expression should be interpreted cautiously when using commercially available antibodies.

Large conductance Ca(2+)-activated K(+) (BK) channels consist of pore-forming α- and accessory ß-subunits. There are four ß-subunit subtypes (ß1-ß4), BK ß1-subunit is specific for smooth muscle cells (SMC). Reduced BK ß1-subunit expression is associated with SMC dysfunction in animal models of human disease, because downregulation of BK ß1-subunit reduces channel activity and increases SMC contractility. Several anti-BK ß1-subunit antibodies are commercially available; however, the specificity of most antibodies has not been tested or confirmed in the tissues from BK ß1-subunit knockout (KO) mice. In this study, we tested the specificity and sensitivity of six commercially available antibodies from five manufacturers. We performed western blot analysis on BK ß1-subunit enriched tissues (mesenteric arteries and colons) and non-SM tissue (cortex of kidney) from wild-type (WT) and BK ß1-KO mice. We found that antibodies either detected protein bands of the appropriate molecular weight in tissues from both WT and BK ß1-KO mice or failed to detect protein bands at the appropriate molecular weight in tissues from WT mice, suggesting that these antibodies may lack specificity for the BK ß1-subunit. The absence of BK ß1-subunit mRNA expression in arteries, colons, and kidneys from BK ß1-KO mice was confirmed by RT-PCR analysis. We conclude that these commercially available antibodies might not be reliable tools for studying BK ß1-subunit expression in murine tissues under the denaturing conditions that we have used. Data obtained using commercially available antibodies should be interpreted cautiously. Our studies underscore the importance of proper negative controls in western blot analyses.

Effect of intravenous amino acids on glutamine and protein kinetics in low-birth-weight preterm infants during the immediate neonatal period.

Glutamine may be a conditionally essential amino acid in low-birth-weight (LBW) preterm neonates. Exogenously administered amino acids, by providing anaplerotic carbon into the tricarboxylic acid cycle, could result in greater cataplerotic efflux and glutamine de novo synthesis. The effect of dose and duration of amino acid infusion on glutamine and nitrogen (N) kinetics was examined in LBW infants in the period immediately after birth. Preterm neonates (<32 weeks gestation, birth weights 809-1,755 g) were randomized to initially receive either 480 or 960 micromol x kg(-1) x h(-1) of an intravenous amino acid solution for 19-24 hours, followed by a higher or lower amino acid load for either 5 h or 24 h. Glutamine de novo synthesis, leucine N, phenylalanine, and urea kinetics were determined using stable isotopic tracers. An increase in amino acid infusion from 480 to 960 micromol x kg(-1) x h(-1) for 5 h resulted in decreased glutamine de novo synthesis in every neonate (384.4 +/- 38.0 to 368.9 +/- 38.2 micromol x kg(-1) x h(-1), P < 0.01) and a lower whole body rate of proteolysis (P < 0.001) and urea synthesis (P < 0.001). However, when the increased amino acid infusion was extended for 24 h, glutamine de novo synthesis increased (369.7 +/- 92.6 to 483.4 +/- 97.5 micromol x kg(-1) x h(-1), P < 0.001), whole body rate of proteolysis did not change, and urea production increased. Decreasing the amino acid load resulted in a decrease in glutamine rate of appearance (R(a)) and leucine N R(a), but had no effect on phenylalanine R(a). Acutely stressed LBW infants responded to an increase in amino acid load by transiently suppressing whole body rate of glutamine synthesis, proteolysis, and oxidation of protein. The mechanisms of this transient effect on whole body protein/nitrogen metabolism remain unknown.

Amino acids, glutamine, and protein metabolism in very low birth weight infants.

Glutamine has been proposed to be conditionally essential for premature infants, and the currently used parenteral nutrient mixtures do not contain glutamine. De novo glutamine synthesis (DGln) is linked to inflow of carbon into and out of the tricarboxylic acid (TCA) cycle. We hypothesized that a higher supply of parenteral amino acids by increasing the influx of amino acid carbon into the TCA cycle will enhance the rate of DGln. Very low birth weight infants were randomized to receive parenteral amino acids either 1.5 g/kg/d for 20 h followed by 3.0 g/kg/d for 5 h (AA1.5) or 3.0 g/kg/d for 20 h followed by 1.5 g/kg/d for 5 h (AA3.0). A third group of babies received amino acids 1.5 g/kg/d for 20 h followed by 3.0 g/kg/d for 20 h (AA-Ext). Glutamine and protein/nitrogen kinetics were examined using [5-(15)N]glutamine, [2H5]phenylalanine, [1-(13)C,15N]leucine, and [15N2]urea tracers. An acute increase in parenteral amino acid infusion for 5 h (AA1.5) resulted in decrease in rate of appearance (Ra) of phenylalanine and urea, but had no effect on glutamine Ra. Infusion of amino acids at 3.0 g/kg/d for 20 h resulted in increase in DGln, leucine transamination, and urea synthesis, but had no effect on Ra phenylalanine (AA-Ext). These data show an acute increase in parenteral amino acid-suppressed proteolysis, however, such an effect was not seen when amino acids were infused for 20 h and resulted in an increase in glutamine synthesis.