Cholesterol secoaldehyde, an ozonation product of cholesterol, induces amyloid aggregation and apoptosis in murine GT1-7 hypothalamic neurons.

Aldehydic products from ozonation of cholesterol and peroxidation of phospholipids have been shown to accelerate aggregation of amyloid-beta (Abeta) in vitro. Here, we show that 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al (ChSeco), an ozonation product of cholesterol, induces Abeta aggregation, generation of reactive oxygen species (ROS), and cytotoxicity in murine GT1-7 hypothalamic neurons. The formation of Abeta aggregates in situ was dose-dependent at ChSeco concentrations ranging from 1 to 20 microM. The increase in insoluble Abeta aggregates at increasing concentrations of ChSeco was accompanied by a decrease in soluble Abeta as evidenced by Western blot analysis. The formation of ROS in neuronal cells was found to be dose- and time-dependent with the magnitude being higher at 20 microM compared to 10 microM ChSeco or untreated controls. The increase in ROS was associated with depletion of GSH. The cytotoxicity induced by ChSeco involved changes in phosphatidylserine translocation, DNA fragmentation, and caspase 3/7 activity that are characteristic of apoptosis. Pretreatment of neuronal cells with Trolox, a water-soluble analog of alpha-tocopherol offered partial, but significant protection against ChSeco-induced cell death, whereas, N-acetyl-L-cysteine (NAC) completely prevented the cytotoxic effects of ChSeco. NAC and Trolox were without any effects on ChSeco-induced Abeta aggregation. Fibrillogenesis inhibitors, which inhibited Abeta aggregation, did not inhibit cell death induced by ChSeco, implying that ROS generation, and not Abeta aggregation, plays a major role in the observed cytotoxicity. However, since Alzheimer's and other neurodegenerative diseases are slow and progressive, the formation of Abeta aggregates in vivo by ChSeco may have long-term pathological consequences.

Amino acids inhibit Agrp gene expression via an mTOR-dependent mechanism.

Metabolic fuels act on hypothalamic neurons to regulate feeding behavior and energy homeostasis, but the signaling mechanisms mediating these effects are not fully clear. Rats placed on a low-protein diet (10% of calories) exhibited increased food intake (P < 0.05) and hypothalamic Agouti-related protein (Agrp) gene expression (P = 0.002). Direct intracerebroventricular injection of either an amino acid mixture (RPMI 1640) or leucine alone (1 mug) suppressed 24-h food intake (P < 0.05), indicating that increasing amino acid concentrations within the brain is sufficient to suppress food intake. To define a cellular mechanism for these direct effects, GT1-7 hypothalamic cells were exposed to low amino acids for 16 h. Decreasing amino acid availability increased Agrp mRNA levels in GT1-7 cells (P < 0.01), and this effect was attenuated by replacement of the amino acid leucine (P < 0.05). Acute exposure to elevated amino acid concentrations increased ribosomal protein S6 kinase phosphorylation via a rapamycin-sensitive mechanism, suggesting that amino acids directly stimulated mammalian target of rapamycin (mTOR) signaling. To test whether mTOR signaling contributes to amino acid inhibition of Agrp gene expression, GT1-7 cells cultured in either low or high amino acids for 16 h and were also treated with rapamcyin (50 nM). Rapamycin treatment increased Agrp mRNA levels in cells exposed to high amino acids (P = 0.01). Taken together, these observations indicate that amino acids can act within the brain to inhibit food intake and that a direct, mTOR-dependent inhibition of Agrp gene expression may contribute to this effect.

Peptide YY and proglucagon mRNA expression patterns and regulation in the gut.

OBJECTIVE: Peptide YY (PYY) and glucagon-like peptide-1 are important in the control of energy homeostasis and are both secreted from the gut in response to ingested nutrients. However, more studies are needed on nutrient regulation of their gene expression patterns in specific areas of the gut. This study detailed PYY and proglucagon (the gene that encodes glucagon-like peptide-1) gene expression patterns and regulation in the gut. We further examined the regulation of PYY and proglucagon mRNA by a diet containing fermentation-resistant starch (in vivo) and butyrate (in vitro). RESEARCH METHODS AND PROCEDURES: Quantitative real time reverse transcriptase-polymerase chain reaction was used to measure PYY and proglucagon gene expression in epithelial cells collected from the duodenum, jejunum, cecum, and colon in normal Sprague-Dawley rats and in rats fed a resistant starch diet for 4 weeks. The same measurements were also performed in primary epithelial cells collected from the cecum and colon of normal rats after the cells were incubated with butyrate for 3 hours. RESULTS: The gene expression patterns for PYY and proglucagon are similar to their peptide distribution patterns in the gut. Also, PYY and proglucagon mRNA expression were up-regulated in the cecum and colon in resistant-starch-fed rats. Butyrate increased PYY and proglucagon gene expression in a dose-dependent manner in vitro. DISCUSSION: Our data provide evidence that the distal part of the gut has the ability to sense nutrients such as butyrate, resulting in the up-regulation of PYY and proglucagon gene expression.

Role of neuronal energy status in the regulation of adenosine 5'-monophosphate-activated protein kinase, orexigenic neuropeptides expression, and feeding behavior.

Nutrient sensing in the hypothalamus is tightly related to food intake regulation. However, the mechanisms by which the nutrient-sensing cells of the brain translate this signal of energy need into feeding behavior via regulation of neuropeptide expression are not known. To address this issue, we investigated two neuronal cell lines expressing agouti-related protein (AgRP), ex vivo hypothalamic tissues, and in vivo whole animals. Maintaining cells in a low cellular ATP concentration generated by low glucose, 2-deoxyglucose (2-DG), ATP synthesis inhibitor, and 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside increased phosphorylation of AMP-activated protein kinase (AMPK) and increased AgRP expression, whereas maintaining cells in high ATP status by high glucose and pyruvate supplementation in 2-DG-treated cells decreased phosphorylation of AMPK and decreased AgRP expression. Overexpression of a dominant-inhibitory mutant of AMPK significantly decreased low-glucose- or 2-DG-induced AgRP expression. Furthermore, ex vivo hypothalamus culture in high glucose concentrations decreased both expression and phosphorylation of AMPK and expression of both AgRP and neuropeptide Y, whereas pyruvate supplementation suppressed a 2-DG-induced AgRP expression. Finally, our in vivo studies clearly show that central administration of pyruvate dramatically delayed 2-DG-induced food intake. These data indicate that modulation of ATP levels in neuronal cells triggers a cascade of events via AMPK that modulate feeding behavior to restore energy status of cells.

Expression levels of genes likely involved in glucose-sensing in the obese Zucker rat brain.

It has been suggested that certain cells in the brain, like pancreatic beta-cells, use glucose transporter-2 (GLUT-2), glucokinase and glucagon-like peptide-1 receptor (GLP-1R) to sense glucose in the service of multiple aspects of energy balance. The obese Zucker rat displays numerous disturbances in energy homeostasis and may provide a model of dysfunctional expression of genes related to nutrient control systems. Using real-time RT-PCR we measured gene expression for three of the pancreatic glucose-sensing markers and neuropeptide Y (NPY) in the medial, lateral hypothalamus and hindbrain of lean and obese Zucker rats of both genders. Additionally, we measured circulating levels of glucose, leptin, insulin, corticosterone and glucagon. The results indicate that GLUT-2 mRNA expression is decreased, whereas glucokinase is increased in the hindbrain of obese rats. NPY mRNA level is significantly higher, whereas GLP-1R is significantly lower in the medial hypothalamus in obese individuals. Gender-related differences were found in the hindbrain and medial hypothalamus for GLUT-2 and in the lateral hypothalamus for GLP-1R and they may be related to the fact that the female Zucker rats do not develop diabetes as readily as males. Furthermore, the hindbrain may be an important site for glucose-sensing where major phenotypic changes occur for glucose-sensing genes expression.

Short-term food restriction and refeeding alter expression of genes likely involved in brain glucosensing.

Several genes involved in glucosensing of the endocrine pancreas have been proposed to serve a similar function in the brain. These genes include the glucose transporter-2 (Glut-2) and glucokinase (GK). In addition, the glucagon-like peptide 1 receptor, which serves as a downstream signal modulator in pancreatic glucosensing and centrally alters feeding, is also of interest. We used quantitative real-time RT-PCR to measure changes in hypothalamic and brainstem Glut-2, GK, and Glp-1R expression of these genes induced by food restriction and refeeding. Sprague-Dawley rats were 50% food restricted for 1 day; one-half of the food-restricted rats were refed with chow for 1 hr before sacrifice. In both hypothalamus and brainstem, gene expression of Glut-2, GK, and Glp-1R was significantly lower in refed rats compared with food-restricted rats. The measures of gene expression in two feeding control groups (ad libitum and voluntarily overfed animals) were intermediate between the food-restricted and refed groups, but were not significantly different from each other. The results indicate that putative glucosensing (GK, Glut-2, and Glp-1R) gene expression in the hypothalamus and brainstem is reduced in response to food intake, depending on prior nutritional status.

Distribution of glucokinase, glucose transporter GLUT2, sulfonylurea receptor-1, glucagon-like peptide-1 receptor and neuropeptide Y messenger RNAs in rat brain by quantitative real time RT-PCR.

Glucokinase (GK), glucose transporter GLUT2, sulfonylurea receptor-1 (SUR1), glucagon-like peptide-1 receptor (GLP-1R) and neuropeptide Y (NPY) have been proposed to be involved in central glucose sensing or regulation of food intake. In this study, we combined tissue micropunch and real time reverse transcription polymerase chain reaction (RT-PCR), and measured GK, GLUT2, SUR1, GLP-1R and NPY mRNA expression in discrete areas in the hypothalamus and the hindbrain.