Fructooligosaccharide feeding during gestation to pregnant mice provided excessive folic acid decreases maternal and female fetal oxidative stress by increasing intestinal microbe-derived hydrogen gas.

Fructooligosaccharide (FOS) is fermented by intestinal microbes to generate intestinal microbe-derived hydrogen gas (IMDH). Oxidative stress increases during gestation, whereas hydrogen gas has antioxidant effects with therapeutic benefits. We have previously reported that the offspring from a pregnant, excessive folic acid mouse model (PEFAM) had abnormal glucose metabolism after growth. We hypothesized that IMDH by FOS feeding during gestation in PEFAM would suppress maternal and fetal oxidative stress. C57BL/6J mice on day 1 of gestation were divided into 3 groups and dissected at gestational day 18. The control (CONT) diet was AIN-93G containing folic acid 2 mg/kg diet; PEFAM was fed with an excessive folic acid (EFA) diet containing folic acid 40 mg/kg diet, and the EFA-FOS diet was replaced half of the sucrose in the EFA diet. Hydrogen gas concentrations in maternal livers and whole fetuses in EFA-FOS were significantly higher than those in CONT and EFA, respectively (P < .05). Maternal and fetal 8-hydroxy-2'-deoxyguanosine in EFA-FOS were not significantly different from those in the CONT group, whereas those in the EFA group were significantly increased compared with CONT and EFA-FOS (P < .05). In EFA-FOS, expression of protein and mRNA of superoxide dismutase and heme oxygenase 1 in mothers and superoxide dismutase in fetuses were not significantly different from those in CONT, whereas those in EFA were significantly increased (P < .05). The protein expression of Nrf2 in mothers and fetuses were not significantly different between EFA-FOS and CONT. Therefore, FOS feeding to PEFAM during gestation decreases maternal and fetal oxidative stress through IMDH.

Excessive folic acid supplementation in pregnant mice impairs insulin secretion and induces the expression of genes associated with fatty liver in their offspring.

OBJECTIVE: Previous human and animal studies have shown that excessive maternal intake of folic acid (FA) predisposes to impaired glucose tolerance in the offspring. However, the underlying mechanism is unknown. Therefore, we aimed to determine whether excessive supplementation with FA during pregnancy affects the glucose tolerance of mouse offspring. RESEARCH METHODS & PROCEDURES: Pregnant C57BL/6J mice were fed AIN93G diet containing either 2 mg [control group (CN)] or 40 mg [high FA group (HFA)] FA/kg diet throughout their pregnancies. On postnatal days (PD)22 and 50, fasting blood glucose was measured in the offspring of both groups, and an oral glucose tolerance test (OGTT) was performed on PD50. On PD53, tissues were collected, and the tissue masses, area of insulin expression in the pancreas, liver triglyceride content, and gene expression were determined. RESULTS: The blood glucose concentrations at 60 and 120 min of the OGTT were higher in female HFA than CN offspring. The serum fasting and non-fasting insulin concentrations and the area of insulin expression in the pancreas were lower in HFA than CN offspring. The liver triglyceride content was higher in female, and tended to be higher in male (P < 0.05), HFA offspring than CN offspring (P < 0.05). The liver mRNA expression of fat synthesis genes, such as Pparγ2 (male and female) and Cidec (male), was higher in HFA than CN offspring (P < 0.05). CONCLUSION: Excessive maternal supplementation of FA in mice leads to lower insulin synthesis and an impairment in hepatic fat metabolism in the offspring.

Neuronal circuits involving neuropeptide Y in hypothalamic arcuate nucleus-mediated feeding regulation.

Neuropeptide Y (NPY) is a 36-amino-acid neuropeptide that was first discovered in porcine brain extracts and later in the porcine intestine. It is widely distributed in both the central and peripheral nervous systems and exerts a powerful orexigenic effect. NPY-producing neuronal cell bodies are abundantly localized in the medial arcuate nucleus of the hypothalamus, this being a brain center that integrates signals for energy homeostasis. Accumulated evidence shows that hypothalamic neuropeptides such as ghrelin, orexin, melanin-concentrating hormone (MCH), galanin-like peptide (GALP) and proopiomelanocortin (POMC) are involved in the regulation of feeding behavior and energy homeostasis via neuronal circuits in the hypothalamus. NPY also forms part of the feeding-regulating neuronal circuitry in conjunction with other feeding-regulating peptide-containing neurons within the hypothalamus. We summarize here current knowledge of the neuronal interactions between NPY and the different types of feeding-regulating peptide-containing neurons in the hypothalamus based on evidence at the immunohistochemicl level and with calcium imaging techniques.

Masked function of amino acid sensors on pancreatic hormone secretion in ventromedial hypothalamic (VMH) lesioned rats with marked hyperinsulinemia.

SUMMARY: In neural regulation of the endocrine pancreas, there is much evidence to suggest that vagal efferents alter insulin and glucagon secretion, but less information on the effects of vagal afferents. In this study, we investigated the role and function of afferent fibers of the vagus nerve in normal and ventromedial hypothalamic (VMH) lesioned rats with marked hyperinsulinemia. In normal rats, hepatic vagotomy was associated with intraperitoneal (ip) arginine-induced enhancement of insulin and glucagon secretion without an accompanying change in blood glucose levels, ip leucine induced enhancement of insulin secretion accompanied by a decrease in blood glucose levels, and ip alanine-induced enhancement of glucagon secretion accompanied by an increase in blood glucose levels. In VMH lesioned rats with marked hyperinsulinemia, none of these amino acids caused significant changes in insulin and glucagon secretion. We conclude that amino acid sensors in normal rats inhibit excess release of pancreatic hormones induced directly by intake of amino acids, such as that in excess protein ingestion, and maintain blood glucose levels within the normal range. In contrast, in VMH lesioned rats with marked hyperinsulinemia, the function of the amino acid sensors is masked due to the marked hyperinsulinemia in these rats.:

Ventromedial hypothalamic lesions enhance small intestinal cell proliferation in mice.

BACKGROUND: We have found previously that ventromedial hypothalamic lesions (VMH) enhance cell proliferation in the visceral organs through vagal hyperactivity in rats. The goal of the current study was to determine the characteristics and nature of cell proliferation in the small intestine in VMH-lesioned mice. METHODS: The weight and length of the small intestine, thickness of the mucosal and muscle layers, number of proliferating cell nuclear antigen (PCNA)-positive cells, and mitotic cell count in the mucosal layer in VMH-lesioned and Sham VMH-lesioned mice were determined at 7 days after the operation. RESULTS: The weight and length of the small intestine in VMH-lesioned mice were significantly greater than those in Sham VMH-lesioned mice, by 11.6% and 15.0%, respectively. The thicknesses of the mucosal and muscle layers of the small intestine in VMH-lesioned mice were also significantly greater than those in Sham VMH-lesioned mice, by 12.7% and 12.5%, respectively. PCNA-positive cells and mitotic cells in the mucosal layer were densely present in crypts in VMH-lesioned mice, and were significantly increased by 31.9% and 71.7%, respectively, compared to Sham VMH-lesioned mice. CONCLUSIONS: These results demonstrate that VMH lesions in mice enhance cell proliferation in the mucosal layers and cause cell hypertrophy or cell proliferation in the muscle layers of the small intestine, which increases the weight and length of the small intestine. VMH lesions in mice may be a new tool for identifying growth factors and related genes involved in enlarging the small intestine mainly through cell proliferation.

Enhanced exercise-induced muscle damage and muscle protein degradation in streptozotocin-induced type 2 diabetic rats.

UNLABELLED: Aims/Introduction: The effects of 5-day voluntary exercise on muscle damage and muscle protein degradation were investigated in a streptozotocin-induced rat model of moderately glycemic, uncontrolled, type 2 diabetes. MATERIALS AND METHODS: In the preliminary experiment, an oral glucose tolerance (1.0 g/kg) test was carried out to confirm the development of diabetes 3 days after streptozotocin treatment (30 mg/kg). In the genuine experiment, rats were divided into four groups: (i) non-diabetic rats without exercise (controls); (ii) non-diabetic rats with exercise; (iii) diabetic rats without exercise; and (iv) diabetic rats with exercise. After 5 days of voluntary wheel running exercise, blood and 24-h urine were collected, and levels of serum creatine kinase, a marker of muscle damage, and 24-h urinary excretion of muscle degradation products were determined. RESULTS: Type 2 diabetic rats with insulin deficiency that exercised had higher serum creatine kinase and greater urinary excretions of creatinine, urea nitrogen and 3-methylhistidine compared with both type 2 diabetic rats with insulin deficiency and non-diabetic rats that did not exercise. However, there were no differences in serum creatine kinase and urinary excretions of creatinine, urea nitrogen and 3-methylhistidine between non-diabetic rats that did and did not exercise. CONCLUSIONS: These findings suggest that muscle damage is induced and muscle protein degradation are enhanced by chronic moderate exercise in moderately glycemic uncontrolled type 2 diabetic rats with insulin deficiency at an intensity level of exercise that does not affect muscle damage and muscle protein degradation in non-diabetic rats. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00130.x, 2011).

Gene expression profiling in rat pancreas after ventromedial hypothalamic lesioning.

OBJECTIVE: We previously reported that vagal hyperactivity produced by ventromedial hypothalamic (VMH) lesions stimulated cell proliferation of rat pancreatic islet B and acinar cells primarily through a cholinergic receptor mechanism. METHODS: This study examined how gene families involved in cell proliferation are regulated after VMH lesions formation. Total pancreatic RNA was extracted, and differences in the gene expression profiles between rats at day 3 after VMH lesioning and sham-VMH-lesioned rats were investigated using DNA microarray and real-time polymerase chain reaction. RESULTS: The VMH lesions regulated the genes that are involved in functions related to cellular growth and proliferation and neuronal development in the pancreas. Real-time polymerase chain reaction also confirmed that gene expressions of angiotensin II receptor-like 1 (AGTRL1) and proline rich 15 (PRR15) were down-regulated at day 3 after the VMH lesions. CONCLUSIONS: Ventromedial hypothalamic lesions may change the expression of cell proliferation-related genes and neuron-related genes in a rat pancreas.

VMH lesions downregulate the expression of Per2 gene in the pancreas in the rat.

The mammalian circadian system contains both central and peripheral oscillators. It was reported that the rhythmic expressions of period homolog 2 (per2) mRNA in peripheral tissues were abolished by the suprachiasmatic nucleus (SCN) lesions. However, there are no reports that ventromedial hypothalamic (VMH) lesions can directly affect the expression of clock genes in pancreas and liver. In the present study, we examined whether VMH lesions can affect the expression of the clock gene, per2 in these organs. Total RNA was extracted from rat pancreas and liver tissues, and differences in the gene expression profiles between rats at day 3 after VMH lesioning and sham-VMH-lesioned rats were investigated using DNA microarray and real-time quantitative analysis. These results showed that VMH lesions downregulated the expression of the clock gene, per2 in the pancreas, but not the liver in the morning. There is a possibility that VMH lesions may affect the expression of the clock gene, per2 in the pancreas.

Gene expression profiling in rat liver after VMH lesioning.

It has been reported that ventromedial hypothalamic (VMH) lesions induce hepatic cell proliferation and apoptosis and metabolic changes in the body. In the present study, we identified genes of which expression profiles showed significant modulation in rat liver after VMH lesions. Total RNA was extracted, and differences in the gene expression profiles between rats at day 3 after VMH lesioning and sham-VMH lesioned rats were investigated using DNA microarray analysis. The results revealed that VMH lesions regulated the genes that were involved in various types of metabolisms and cell proliferations in the liver. Real-time PCR also confirmed that gene expressions of ELOVL6 and SPC24 were upregulated, and that of SERPINA7 was downregulated. VMH lesions may change the expressions of multiple metabolism genes and cell proliferation-related genes in rat liver.

Ventromedial hypothalamic lesions change the expression of neuron-related genes and immune-related genes in rat liver.

There are no reports that hypothalamus can directly affect the expression of neuron-related genes and immune-related genes in liver. We identified genes of which expression profiles showed significant modulation in rat liver after ventromedial hypothalamic (VMH) lesions. Total RNA was extracted, and differences in the gene expression profiles between rats at day 3 after VMH lesioning and sham-VMH lesioned rats were investigated using DNA microarray analysis. The result revealed that VMH lesions regulated the genes that were involved in functions related to neuronal development and immunofunction in the liver. Real-time PCR also confirmed that gene expression of SULT4A1 was upregulated, but expression of ACSL1 and CISH were downregulated at day 3 after VMH lesions. VMH lesions may change the expression of neuron-related genes and immune-related genes in rat liver.

Effects of gastric vagotomy on visceral cell proliferation induced by ventromedial hypothalamic lesions: role of vagal hyperactivity.

In rats, ventromedial hypothalamic (VMH) lesions induce cell proliferation in the visceral organs (stomach, small intestine, liver, and pancreas) due to hyperactivity of the vagus nerve. To investigate the effects of selective gastric vagotomy on VMH lesion-induced cell proliferation and secretion of gastric acid, we assessed the mitotic index (the number of proliferating cell nuclear antigen (PCNA)-immunopositive cells per 1,000 cells in the gastric mucosal cell layer) and measured the volume of secreted basal gastric acid. Furthermore, to explore whether or not ethanol-induced acute gastric mucosal lesions (AGML) lead to ulcer formation in VMH-lesioned rats, we assessed the ulcer index of both sham-operated and VMH-lesioned rats after administration of ethanol. VMH lesions resulted in an increased mitotic index and thickness of the gastric mucosal cell layer and gave rise to the hypersecretion of gastric acid. Selective gastric vagotomy restored these parameters to normal without affecting cell proliferation in other visceral organs. Ethanol-induced AGML caused ulcers in sham VMH-lesioned rats, whereas VMH-lesioned rats were less likely to exhibit such ulcers. These results suggest that VMH lesion-induced vagally mediated cell proliferation in the visceral organs is associated with hyperfunction in these organs, and VMH lesion-induced resistance to ethanol may be due to thickening of the gastric mucosal cell layer resulting from cell proliferation in the gastric mucosa-this in turn is due to hyperactivity of the vagus nerve.

Changes of neuron-specific and apoptosis gene expression levels after ventromedial hypothalamic lesions in rat intestine.

The intestinal epithelium is continuously renewed through a balance between cell proliferation and apoptosis. We identified genes of which expression profiles showed significant modulation, and we investigated the cellular mechanisms of this gene regulation in rat intestine after ventromedial hypothalamic (VMH) lesions. Total RNA was extracted, and differences in the gene expression profiles between rats at day 3 after VMH lesioning and in sham-VMH lesioned rats were investigated using DNA microarray analysis and real-time polymerase chain reaction (PCR) methods. DNA microarray analysis revealed that VMH lesions regulated the genes that were involved in functions predominantly related to neuronal development, cell proliferation and apoptosis. Real-time PCR also confirmed that gene expressions of Efnb1 were downregulated. Meanwhile, expression of Casp3 was similar. It is noted that the signaling networks of many gene families, including neuron-specific genes and apoptosis genes in the intestine were changed after VMH lesioning. VMH lesions may suppress mainly the caspase independent type II pathway for apoptosis and induce cell proliferation in the intestine.

Visualization of ghrelin-producing neurons in the hypothalamic arcuate nucleus using ghrelin-EGFP transgenic mice.

The gut-brain hormone ghrelin is known to stimulate growth hormone release from the pituitary gland, and to regulate appetite and energy metabolism. Ghrelin-containing neurons have been shown to form neuronal network with several types of appetite-regulating neurons in the hypothalamus. Although ghrelin-containing cell bodies have been reported to localize in the hypothalamic arcuate nucleus, the published results present large discrepancies regarding the localization of ghrelin-positive cell bodies in the brain. In order to address this issue, we have generated a transgenic mouse model by microinjecting a DNA construct in which the transcription regulatory regions of ghrelin drive the enhanced green fluorescent protein (EGFP) gene. These transgenic mice expressed EGFP and ghrelin mRNA in the stomach and hypothalamus. Double immunostaining revealed that GFP-like immunoreactivity was co-localized with ghrelin-like immunoreactivity in the stomach of these animals, while EGFP fluorescence was clearly demonstrated in the hypothalamic arcuate nucleus by confocal laser microscopy. The ghrelin-EGFP transgenic mouse model described in this study therefore provides a powerful tool with which to analyze ghrelin neuronal circuits in the brain and should contribute to our understanding of the functional significance of ghrelin in the central nervous system.

Synaptic relationships between proopiomelanocortin- and ghrelin-containing neurons in the rat arcuate nucleus.

Both proopiomelanocortin (POMC) and ghrelin peptides are implicated in the feeding regulation. The synaptic relationships between POMC- and ghrelin-containing neurons in the hypothalamic arcuate nucleus were studied using double-immunostaining methods at the light and electron microscope levels. Many POMC-like immunoreactive axon terminals were found to be apposed to ghrelin-like immunoreactive neurons and also to make synapses with ghrelin-like immunoreactive neuronal perikarya and dendritic processes. Most of the synapses were symmetrical in shape. A small number of synapses made by ghrelin-like immunoreactive axon terminals on POMC-like immunoreactive neurons were also identified. Both the POMC- and ghrelin-like immunoreactive neurons were found to contain large dense granular vesicles. These data suggest that the POMC-producing neurons are modulated via synaptic communication with ghrelin-containing neurons. Moreover, ghrelin-containing neurons may also have a feedback effect on POMC-containing neurons through direct synaptic contacts.

Neuropeptide W is expressed in the noradrenalin-containing cells in the rat adrenal medulla.

Neuropeptide W (NPW) is an endogenous ligand for GPR7, a member of the G-protein-coupled receptor family. NPW plays an important role in the regulation of both feeding and energy metabolism, and is also implicated in modulating responses to an acute inflammatory pain through activation of the hypothalamus-pituitary-adrenal axis. GPR7 mRNA has been shown to be expressed in the hypothalamus, pituitary gland and adrenal cortex. Similarly, NPW expression has been demonstrated in the brain and pituitary gland. However, the precise distribution of NPW-producing cells in the adrenal gland remains unknown. The aim of this study was to explore the distribution and localization of NPW immunoreactivity in the rat adrenal gland. Total RNA was prepared from the hypothalamus, pituitary gland and adrenal gland. RT-PCR revealed the expression of NPW mRNA in these tissues, while in situ hybridization demonstrated the presence of NPW mRNA in the adrenal medulla. When immunohistochemistry was performed on sections of adrenal gland, NPW-like immunoreactivity (NPW-LI) was observed in the medulla but not in the cortex. Moreover, NPW-LI was found to be co-localized in cells which expressed dopamine beta hydroxylase but not phenylethanolamine-N-methyltransferase. The finding that NPW is expressed in noradrenalin-containing cells in the adrenal medulla suggests that it may play an important role in endocrine function in the adrenal gland.

Neural interaction between galanin-like peptide (GALP)- and luteinizing hormone-releasing hormone (LHRH)-containing neurons.

Galanin-like peptide (GALP), commonly known as an appetite-regulating peptide, has been shown to increase plasma luteinizing hormone (LH) through luteinizing hormone-releasing hormone (LHRH). This led us to investigate, using both light and electron microscopy, whether GALP-containing neurons in the rat brain make direct inputs to LHRH-containing neurons. As LHRH-containing neurons are very difficult to demonstrate immunohistochemically with LHRH antiserum without colchicine treatment, we used a transgenic rat in which LHRH tagged with enhanced green fluorescence protein facilitated the precise detection of LHRH-producing neuronal cell bodies and processes. This is the first study to report on synaptic inputs to LHRH-containing neurons at the ultrastructural level using this transgenic model. We also used immunohistochemistry to investigate the neuronal interaction between GALP- and LHRH-containing neurons. The experiments revealed that GALP-containing nerve terminals lie in close apposition with LHRH-containing cell bodies and processes in the medial preoptic area and the bed nucleus of the stria terminalis. At the ultrastructural level, the GALP-positive nerve terminals were found to make axo-somatic and axo-dendritic synaptic contacts with the EGFP-positive neurons in these areas. These results strongly suggest that GALP-containing neurons provide direct input to LHRH-containing neurons and that GALP plays a crucial role in the regulation of LH secretion via LHRH.