Three weeks voluntary running wheel exercise increases endoplasmic reticulum stress in the brain of mice.

The unfolded protein response (UPR) is a dynamic cellular mechanism for reducing endoplasmic reticulum (ER) stress. ER stress occurs from a variety of causes such as nutritional deprivation or over-nutrition, expression of misfolded or mutant proteins and increased synthesis of secretory protein. Obesity induced by over-nutrition has been associated with ER stress. Although exercise has a beneficial effect in opposing the development of obesity and neurodegenerative diseases, there have been no studies on the effect of exercise on ER stress in the brain induced by over-nutrition. We have taken advantage of the substantial individual differences in voluntary running activity among inbred C57BL/6 mice to investigate the relation between ER stress within regions of the brain and voluntary running activity in mice fed on either a low fat or high fat diet while maintained individually in cages with running wheels. Mice were divided into three groups depending on their voluntary running level and compared with a sedentary group. ER stress was assayed by real-time PCR and Western blots of the UPR pathway markers Xbp1, PERK, eIF2alpha, Hspa5 and ATF6. Three weeks of HFD had little effect on ER stress in the brain of the sedentary group compared to animals fed the LFD. Higher voluntary running activity was associated with increased ER stress in the hypothalamus, hippocampus and cortex. The responses were largest in the hypothalamus. The increase in the UPR response in response to exercise did not induce apoptotic signals and may thus contribute to the protective effect of exercise in preventing neurodegenerative disease.

Melanocortin activity in the amygdala controls appetite for dietary fat.

The amygdala is rich in melanocortin 4 receptors. Because the reduction in dietary fat intake after enterostatin is injected in the central nucleus of the amygdala (CeA) is blocked by a melanocortin 4 receptor antagonist, we investigated the role of melanocortin activity in the CeA in regulating food intake and macronutrient choice. Sprague-Dawley rats, fitted with CeA cannulas, were fed either chow, a high-fat (HF) diet, or adapted to a two-choice HF or low-fat (LF) diet. Injections of the MC4R agonist melanotan II (MTII) in the CeA had a dose-dependent inhibitory effect on food intake that lasted for at least 24 h. This response was greater in rats fed a HF diet. The inverse agonist agouti-related protein (AgRP) and antagonist SHU-9119 increased food intake in a dose-dependent manner, with the hyperphagia lasting for 60 h. In rats adapted to a two-choice HF/LF diet, MTII decreased HF consumption but had no effect on LF consumption, resulting in a long-lasting decrease in total calorie intake (-35.5% after 24 h, P < 0.05). Total calorie intake increased in both AgRP- and SHU-9119-treated rats (32 and 109% after 24 h, respectively) as the result of increased intake of HF diet. There was no modification of LF consumption with AgRP treatment and a transient nonsignificant decrease with SHU-9119 treatment. Amygdala brain-derived neurotrophic factor expression was increased by AgRP in fed rats. These results identify the amygdala as a site of action for the melanocortin system to control food intake and dietary preferences.

High-fat diets induce a rapid loss of the insulin anorectic response in the amygdala.

Intracerebroventricular insulin decreases food intake (FI). The central bed nucleus of the amygdala (CeA), as other regions of the brain regulating feeding behavior, expresses insulin receptors. Our objectives were to show an insulin anorectic response in the amygdala, study the effect of high-fat diets on this response, and map the neural network activated by CeA insulin using c-Fos immunohistochemistry. Sprague-Dawley (SD) rats fitted with unilateral CeA cannulas were adapted to a low-fat (LFD) diet before they were fed a high-fat diet (HFD). Their feeding response to CeA saline or insulin (8 mU) was tested after 24 h, 72 h, or 7 days of being on a HFD. In a second experiment, SD rats were fed the HFD for 3, 7, or 49 days and were then refed with the LFD. They were tested for their insulin response before and after an HFD and every 3 days for the following weeks. Insulin tolerance tests were performed in a parallel group of rats. The CeA insulin stimulation c-Fos expression was studied to identify the distribution of activated neuronal populations. Feeding an HFD for 72 h or more induced a CeA, but not peripheral, insulin resistance, which was slowly reversed by LFD refeeding. The duration of HFD feeding determined the time frame for reversal of the insulin resistance. CeA insulin increased c-Fos in multiple brain regions, including the arcuate nucleus/paraventricular nucleus region of the hypothalamus. We conclude that the amygdala may be an important site for insulin regulation of food intake and may have a significant role in determining susceptibility to HFD-induced obesity.

Enterostatin alters protein trafficking to inhibit insulin secretion in Beta-TC6 cells.

Enterostatin is a peptide that regulates dietary fat intake in rodents and inhibits insulin secretion from pancreatic beta cells. Microarray studies of the genomic response of both a human hepatoma cell line (HepG2 cells) and a mouse hypothalamic cell line (GT1-7 cells) to enterostatin suggested that it might regulate protein trafficking. Using semi-quantitative real-time PCR and Western blot analysis, we confirmed that enterostatin upregulated Scamp2 and down regulated Dynamin2 in these cell lines. The receptor for enterostatin is the F1-ATPase beta subunit. We transfected HepG2 cells with either a green fluorescent protein (GFP) tagged F1-ATPase beta subunit or a red fluorescent protein (RFP) tagged F1-ATPase alpha subunit to study the effects of enterostatin on translocation of its own receptor protein. Enterostatin induced movement of GFP-beta subunit to the cell periphery area but did not have any effect on the localization of RFP-alpha subunit protein in HepG2. As Scamp2 is involved in glucose uptake in mouse Beta-TC6 insulinoma cells we tested enterostatin's effect in Beta-TC6 cells. Glucose stimulated insulin release was inhibited by enterostatin as reported previously. Using siRNA to Scamp2 did not change glucose stimulated insulin release but siRNA to Dynamin2 and dominant negative Dynamin2 (Dyn K44A) inhibited glucose stimulated insulin release and abolished the response to enterostatin. This suggests enterostatin inhibits glucose stimulated insulin release in pancreatic beta cells through down regulation of Dynamin2. This study also suggests that enterostatin might have a more generalized effect on protein trafficking in various cells.

Enterostatin affects cyclic AMP and ERK signaling pathways to regulate Agouti-related protein (AgRP) expression.

Enterostatin, a gut-brain peptide, inhibits dietary fat intake in rats. The purpose of this study was to identify the intracellular signaling pathways that are responsive to enterostatin and that modulate the effects of enterostatin on the expression of Agouti-related protein (AgRP). We used the hypothalamic GT1-7 neuronal cell line to identify the effects of enterostatin on cyclic AMP and ERK signaling using conventional immunoassays or Western blots to assay the activity of these pathways. Enterostatin enhanced the level of cyclic AMP, PKA(RIIbeta) and phospho-CREB and increased pERK levels in GT 1-7 cells. The effects on pERK were rapid (7.5 min) and dose-dependent. These signaling responses were blocked by an antibody to the enterostatin receptor (beta subunit of F1-ATPase), by the pERK inhibitor U0126 and by the P2Y receptor antagonist Suramin. Enterostatin showed a biphasic effect on AgRP mRNA, initially increasing but subsequently decreasing the levels. The cyclic AMP activator Sp-cAMP increased AgRP mRNA expression. Transfection of a wild type ERK construct reduced AgRP mRNA levels. Enterostatin inhibited expression of Krüppel-like factor 4 (KLF4), a transcriptional regulator of AgRP. KLF4 gene expression was increased by Sp-cAMP but decreased by wild-type ERK expression. U0126 blocked the effect of enterostatin on KLF4 expression. We conclude that enterostatin binding to its receptor activates the pERK pathway to inhibit AgRP gene expression but may enhance AgRP expression through activation of the cyclic AMP pathway. These pathways probably mediate the enterostatin inhibition of dietary fat intake.

Enterostatin inhibition of dietary fat intake is modulated through the melanocortin system.

Enterostatin injected into the amygdala selectively reduces dietary fat intake by an action that involves a serotonergic component in the paraventricular nucleus. We have investigated the role of melanocortin signaling in the response to enterostatin by studies in melanocortin 4 receptor (MC4R) knock out mice and by the use of the MC4R and MC3R antagonist SHU9119, and by neurochemical phenotyping of enterostatin activated cells. We also determined the effect of enterostatin in vivo on the expression of AgRP in the hypothalamus and amygdala of rats and in culture on a GT1-7 neuronal cell line. Enterostatin had no effect on food intake in MC4R knock out mice. SHU9119 i.c.v. blocked the feeding response to amygdala enterostatin in rats. Amygdala enterostatin induced fos activation in alpha-melanocyte stimulating hormone (alpha-MSH) neurons in the arcuate nucleus. Enterostatin also reduced the expression of AgRP in the hypothalamus and amygdala and in GT1-7 cells. These data suggest enterostatin inhibits dietary fat intake through a melanocortin signaling pathway.

Fatty acids and glucose in high concentration down-regulates ATP synthase beta-subunit protein expression in INS-1 cells.

Chronic hyperglycemia and hyperlipidemia exert deleterious effects on beta-cell function and impair glucose-induced insulin release, referred to as glucotoxicity and lipotoxticity. These abnormalities are associated with decreased glucose-induced ATP production; ATP serves as an important signal for insulin secretion. To investigate the mechanism of the impaired ATP formation, we examined the effects of elevated glucose and fatty acids levels on ATP synthase beta-subunit expression, ATP content and insulin secretion in INS-1 insulinoma beta-cells. ATP synthase beta-subunit expression was measured by western blot, ATP content was monitored by ATP luminescence and insulin secretion detected by radio immunoassay. Our result indicated that chronic exposure to high doses of fatty acids together with high levels glucose produced a marked decrease in ATP synthase beta-subunit protein expression. Reduction of ATP synthase beta-subunit protein expression occurred with a decreased intracellular ATP concentration and insulin secretion at high fatty acid concentrations. These results indicate that high glucose together with fatty acids impair the production of ATP in beta-cells through the suppression of mitochondrial ATP synthesis. We conclude that ATP synthase beta-subunit may have an important role in the glucolipotoxicity of islet cells and suggest that ATP synthase beta-subunit might be a target of lipotoxicity in beta-cells.

Procolipase gene expression in the rat brain: source of endogenous enterostatin production in the brain.

Enterostatin is a pentapeptide released from its precursor protein procolipase, which is synthesized in the exocrine pancreas and gastric mucosa. As central injection of enterostatin has potent effects on feeding, we hypothesized that the procolipase may also be expressed in the brain. We confirmed the presence of preprocolipase gene expression in amygdala by reverse transcription-polymerase chain reaction and Northern blot analysis and of protein expression by Western blots. Immunohistochemical analysis using antibodies for procolipase and enterostatin identified their immunoreactivity (IR) in rat brain. Procolipase IR was present in the cytoplasm of paraventricular, amygdala, and the dorsal thalamus nucleus. Enterostatin IR was evident in the fibers of the dorsal thalamus and arcuate nucleus. In vivo injection of enterostatin antibody into rat amygdala increased food intake. These data suggest that procolipase and enterostatin are synthesized within specific regions of the brain that function in the regulation of food intake centrally.

Different metabolic responses to central and peripheral injection of enterostatin.

Enterostatin, a pentapeptide cleaved from procolipase, suppresses fat intake after peripheral and central administration. Chronic treatment of rats with enterostatin decreases body weight and body fat. The effect was greater than could be accounted by the reduction in food intake alone. Hence, we have investigated the effect of enterostatin on energy metabolism. Male Sprague-Dawley rats adapted to a high-fat diet were implanted with lateral cerebral ventricular or amygdala cannulas. The metabolic effects were determined by indirect calorimetry. After habituation to the test cages, fasted rats were injected with either saline vehicle or enterostatin given either intraperitoneally (100 nmol) or intracerebroventricularly (1 nmol) or into specific brain regions [amygdala (0.01 nmol) or paraventricular nucleus (PVN) (0.1 nmol)]. Respiratory quotient (RQ) and energy expenditure were monitored over 2 h. Intraperitoneal enterostatin reduced RQ (saline: 0.81 +/- 0.02 vs. enterostatin: 0.76 +/- 0.01) and increased energy expenditure by 44%. Intracerebroventricular enterostatin increased the energy expenditure without any effects on RQ, whereas PVN enterostatin increased metabolic rate, while preventing the increase in RQ observed in the control animals. In contrast, neither RQ nor energy expenditure was altered after enterostatin was injected into the amygdala. Enterostatin activated AMP-activated protein kinase in primary cultures of human myocytes in a dose- and time-dependent manner and increased the rate of fatty acid beta-oxidation. These findings suggest that enterostatin regulates energy expenditure and substrate partitioning through both peripheral and central effects.

Leucine regulation of glucokinase and ATP synthase sensitizes glucose-induced insulin secretion in pancreatic beta-cells.

We have recently shown that leucine culture upregulates ATP synthase beta-subunit (ATPSbeta) and increases ATP level, cytosolic Ca(2+), and glucose-induced insulin secretion in rat islets. The aim is to test whether glucokinase expression is also affected in rat islets and its role in glucose sensitization during leucine culture. Leucine culture increased glucose-induced NAD(P)H level at 1 and 2 days but not at 1 week. The half-maximal effective concentration of the glucose response curve for NAD(P)H was left-shifted from 5-7 to 2-3 mmol/l. The effect was dose dependent and rapamycin insensitive. Leucine culture did not affect glyceraldehyde effects on NAD(P)H. Leucine pretreatment for 30 min had no effects on NAD(P)H levels. Leucine culture for 2 days also increased glucose-induced cytosolic Ca(2+) elevation, ATP level, and insulin secretion. Leucine increase of glucokinase mRNA levels occurred as early as day 1 and lasted through 1 week. That of ATPSbeta did not occur until day 2 and lasted through 1 week. Leucine effects on both mRNAs were dose dependent. The upregulation of both genes was confirmed by Western blotting. Leucine culture also increased glucose-induced insulin secretion, ATP level, glucokinase, and ATPSbeta levels of type 2 diabetic human islets. In conclusion, leucine culture upregulates glucokinase, which increases NAD(P)H level, and ATPSbeta, which increases oxidation of NADH and production of ATP. The combined upregulation of both genes increases glucose-induced cytosolic Ca(2+) and insulin secretion.

Extensive diversity of ionizing-radiation-resistant bacteria recovered from Sonoran Desert soil and description of nine new species of the genus Deinococcus obtained from a single soil sample.

The ionizing-radiation-resistant fractions of two soil bacterial communities were investigated by exposing an arid soil from the Sonoran Desert and a nonarid soil from a Louisiana forest to various doses of ionizing radiation using a (60)Co source. The numbers of surviving bacteria decreased as the dose of gamma radiation to which the soils were exposed increased. Bacterial isolates surviving doses of 30 kGy were recovered from the Sonoran Desert soil, while no isolates were recovered from the nonarid forest soil after exposure to doses greater than 13 kGy. The phylogenetic diversities of the surviving culturable bacteria were compared for the two soils using 16S rRNA gene sequence analysis. In addition to a bacterial population that was more resistant to higher doses of ionizing radiation, the diversity of the isolates was greater in the arid soil. The taxonomic diversity of the isolates recovered was found to decrease as the level of ionizing-radiation exposure increased. Bacterial isolates of the genera Deinococcus, Geodermatophilus, and Hymenobacter were still recovered from the arid soil after exposure to doses of 17 to 30 kGy. The recovery of large numbers of extremely ionizing-radiation-resistant bacteria from an arid soil and not from a nonarid soil provides further ecological support for the hypothesis that the ionizing-radiation resistance phenotype is a consequence of the evolution of other DNA repair systems that protect cells against commonly encountered environmental stressors, such as desiccation. The diverse group of bacterial strains isolated from the arid soil sample included 60 Deinococcus strains, the characterization of which revealed nine novel species of this genus.

The F1-ATPase beta-subunit is the putative enterostatin receptor.

It has been suggested that the F1-ATPase beta-subunit is the enterostatin receptor. We investigated the binding activity of the purified protein with a labeled antagonist, beta-casomorphin1-7, in the absence and presence of cold enterostatin. 125I-beta-casomorphin1-7 weakly binds to the rat F1-ATPase beta-subunit. Binding was promoted by low concentrations of cold enterostatin but displaced by higher concentrations. To study the relationship between binding activity and feeding behavior, we examined the ability of a number of enterostatin analogs to affect beta-casomorphin1-7 binding to the F1-ATPase beta-subunit. Peptides that suppressed food intake promoted beta-casomorphin1-7 binding whereas peptides that stimulated food intake or did not affect the food intake displaced beta-casomorphin1-7 binding. Surface plasmon resonance measurements show that the beta-subunit of F1-ATPase binds immobilized enterostatin with a dissociation constant of 150 nM, where no binding could be detected for the assembled F1-ATPase complex. Western blot analysis showed the F1-ATPase beta-subunit was present on plasma and mitochondrial membranes of rat liver and amygdala. The data provides evidence that the F1-ATPase beta-subunit is the enterostatin receptor and suggests that enterostatin and beta-casomorphin1-7 bind to distinct sites on the protein.

Analysis of Deinococcus radiodurans's transcriptional response to ionizing radiation and desiccation reveals novel proteins that contribute to extreme radioresistance.

During the first hour after a sublethal dose of ionizing radiation, 72 genes were upregulated threefold or higher in D. radiodurans R1. Thirty-three of these loci were also among a set of 73 genes expressed in R1 cultures recovering from desiccation. The five transcripts most highly induced in response to each stress are the same and encode proteins of unknown function. The genes (ddrA, ddrB, ddrC, ddrD, and pprA) corresponding to these transcripts were deleted, both alone and in all possible two-way combinations. Characterization of the mutant strains defines three epistasis groups that reflect different cellular responses to ionizing radiation-induced damage. The ddrA and ddrB gene products have complementary activities and inactivating both loci generates a strain that is more sensitive to ionizing radiation than strains in which either single gene has been deleted. These proteins appear to mediate efficient RecA-independent processes connected to ionizing radiation resistance. The pprA gene product is not necessary for homologous recombination during natural transformation, but nevertheless may participate in a RecA-dependent process during recovery from radiation damage. These characterizations clearly demonstrate that novel mechanisms significantly contribute to the ionizing radiation resistance in D. radiodurans.