Browsing by Subject "Ghrelin"
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Item Confirmation of Hypoglycemia in Goat -/- Mice When Total Body Fat Falls below 2% of Body Weight(2013-01-22) Singh, Ashish; Goldstein, Joseph L.; Zhao, Tongjin; Brown, Michael S.Ghrelin is an octanoylated peptide hormone first identified in stomach, with the octanoyl group being essential to its biological activity. The enzyme that attaches the octanoyl group to ghrelin is called Ghrelin-Oacyltransferase (GOAT). By studying mice that have the GOAT gene knocked out (GOAT KO mice), we have shown that these mice develop severe hypoglycemia under a 60% calorie restricted diet. In order for this hypoglycemia to occur, depletion of fat deposits is required. Specifically, GOAT knockout mice will not develop severe hypoglycemia until the total fat mass drops to 2% of the total body weight. These observations were made in 8-week-old mice with an average starting fat mass between 8-10% of total body weight. In our present work, we wanted to know whether we could reproduce the results using older mice with a higher percentage of fat mass. The mice used in this study were 32-34 week old male mice (wild type and GOAT knockout mice, n=8/group), and both groups had an average starting fat mass of 17% of total body weight. We then subjected these mice to a 60% calorie restriction and monitored their fat mass and blood glucose level everyone or two days. For the first 7 days of calorie restriction, both wild type and GOAT knockout mice were able to maintain their blood glucose around 60 mg/dl. After that, the GOAT knockout mice start to develop hypoglycemia when their body fat mass dropped below 2% of the body weight. However, the wild type mice were able to maintain their blood glucose level above 40 mg/dl throughout the course even when their fat mass dropped below 2% of their body weight. The results here further confirm that in order to develop hypoglycemia in the GOAT knockout mice, the fat mass needs to be depleted from these mice during calorie restriction , even in older mice (32-34 weeks versus 8 weeks).Item Control of appetite in mouse and man by the hormone ghrelin(2013-04-05) Zigman, Jeffrey M.Item Essential Function of Ghrelin in Chronic Starvation(2013-02-22) Li, Robert Lin; Elmquist, Joel; Brown, Michael S.; Goldstein, Joseph L.; Cobb, Melanie H.; Kliewer, Steven A.Ghrelin, an octanoylated peptide hormone secreted from the stomach, stimulates the release of growth hormone (GH) from the pituitary. Ghrelin O-acyltransferase (GOAT) is the enzyme required for the attachment of octanoate to serine-3 of ghrelin, a step essential for making active ghrelin. In this study, we eliminated the Goat gene from mice to produce Goat –/– mice that lack octanoylated ghrelin. These mice were indistinguishable in weight from their wild-type (WT) littermates in when fed either a normal or a high fat diet. On 60% calorie restriction, WT and Goat –/– mice lost 30% of their body weight and 75% of their body fat within the first 4 days. While fasting blood glucose levels declined at the same rate initially in WT and Goat –/– mice, levels in the WT mice stabilized at 58–76 mg/dL after 4 days of 60% calorie restriction. In contrast, fasting blood glucose levels in the calorie restricted Goat –/– mice continued declining to 12–36 mg/dL by day 7, at which point the mice were moribund. Levels of ghrelin and GH rose progressively in WT mice during the calorie restriction. GH levels in Goat –/– mice, which have no ghrelin, rose to a much lesser degree, a phenotype also seen in calorie restricted Preproghrelin –/– mice that lack both ghrelin and des-acyl ghrelin. Restoring ghrelin or GH via an osmotic minipump to calorie restricted Goat –/– mice rescued their hypoglycemia. Thus, ghrelin is essential for survival during severe calorie restriction by elevating GH levels to preserve blood glucose and maintain life. The decreased elevation of GH in calorie restricted Goat –/– mice was associated with decreased plasma levels of two gluconeogenic substrates: pyruvate and lactate. Injections of exogenous pyruvate, lactate, and alanine to calorie restricted Goat –/– mice prevented the development of hypoglycemia. Injections of exogenous octanoate to calorie restricted Goat –/– mice, which spares the need to oxidize glucose and gluconeogenic substrate in the tricarboxylic (TCA) cycle to provide energy for gluconeogenesis, also prevented the hypoglycemia. Therefore, the preservation of blood glucose during calorie restriction by the ghrelin-mediated rise in GH involves the maintenance of adequate plasma levels of gluconeogenic substrates. The dramatic rise in plasma ghrelin during chronic severe calorie deprivation is essential to maintain life. However, the mechanism for this increase is not understood. From tissue culture cells derived from mice bearing ghrelinomas induced by a tissue-specific SV40 T-antigen transgene, we found that ghrelin secreting cells express high levels of mRNA encoding the β1-adrenergic receptor. Ghrelin secretion from these cells was stimulated by the addition of norepinephrine or epinephrine, an effect blocked by atenolol, a selective β1-adrenergic antagonist. Treating WT mice with atenolol or reserpine, a drug that depletes adrenergic neurotransmitters from sympathetic neurons, blocked the fasting-induced increase in plasma ghrelin. Thus, ghrelin secretion during fasting is induced by adrenergic agents released by sympathetic neurons which act directly on β1 receptors on the ghrelin-secreting cells of the stomach.Item From Feast to Famine: A Tale of Satiety and Hunger Hormones(2016-04-18) Zhang, Yuanyuan; Repa, Joyce J.; Horton, Jay D.; Chen, Zhijian J.; Goldstein, Joseph L.; Brown, Michael S.Ghrelin is a peptide hormone secreted mainly from the stomach. It has a unique octanoylation on Ser-3 by Ghrelin-O-Acyltransferase (GOAT). We have previously shown that Goat−/− mice developed severe hypoglycemia under 60% calorie restriction. Liver autophagy has been reported to play a crucial role in maintaining blood glucose during fasting. The present work was carried out to explore whether autophagy plays a role in the onset of hypoglycemia in Goat−/− mice. We observed a deficiency in autophagy in livers of calorie-restricted Goat−/− mice by showing lower expression level of LC3-II, an autophagy marker. This was further demonstrated by showing 10-fold fewer autolysosomes in livers of calorie-restricted Goat−/− mice as compared to the control mice (20 electron microscopic images analyzed for each group). We then went on to show that the deficiency in autophagy in Goat−/− mice can be restored by infusion of growth hormone. It can also be restored by injections of lactate, a gluconeogenic precursor, or octanoate, a fatty acid that spares the usage of glucose. Protein expression of p- STAT 5, a downstream target of growth hormone action, was significantly lower in livers of calorie-restricted Goat−/− mice, and was restored by infusion of growth hormone and by injections of lactate or octanoate. Protein expression levels of LC3-II and p-STAT 5 showed a strong correlation (r2=0.87, p<10-6) through the time course of calorie-restriction. Considered together, these data suggest that the onset of autophagy during calorie restriction is strongly correlated with the ghrelin-growth hormone axis, and that autophagy plays an important role in maintaining blood glucose homeostasis during chronic starvation.Item Ghrelin: The Hunger Hormone that Isn't(2014-06-09) McFarlane, Matthew Ryan; Elmquist, Joel; Brown, Michael S.; Goldstein, Joseph L.; Olson, Eric N.; Horton, Jay D.Ghrelin is a 28-amino acid acylated peptide hormone secreted by endocrine cells in the stomach. It was first identified in 1999 and shortly thereafter shown to stimulate appetite when injected into rodents and humans. While ghrelin knockout mice have failed to show a decrease in appetite or bodyweight, the literature -- as well as the lay press -- continues to presume ghrelin levels are a mediator of appetite in vivo. For example, the suppression of ghrelin levels secondary to gastric bypass surgery is frequently invoked as a contributing factor in the resulting weight loss. In the literature, this incongruity has been rationalized as embryonic or neonatal compensation, a claim predicated on a study by Luquet et al. which showed that AgRP/NPY neurons (which express the ghrelin receptor and are thought to be the critical target for appetite stimulation) can be ablated without consequence in neonatal mice, while in adult mice ablation causes a rapid and profound loss of appetite. Widespread acceptance notwithstanding, the hypothesis that a reduction in ghrelin levels decreases appetite in adults has never been tested. We generated a mouse line expressing the simian diphtheria toxin receptor on ghrelin cells. With these mice we are able to rapidly ablate ghrelin cells in adulthood with the injection of diphtheria toxin. Despite an 80-95% loss of circulating ghrelin, our mice show no decrease in appetite or body weight in the short or long term and become obese and hyperinsulinemic in response to high fat feeding. To investigate why ghrelin seems to be sufficient but not necessary for hunger, we injected increasing doses of ghrelin and measured both food intake and the resulting plasma concentration. We found that the threshold dose for an appetite response raised blood concentrations more than 50-fold above physiologic levels -- well above the highest concentration we have observed even during extreme starvation. We show that at physiologic levels ghrelin is neither necessary nor sufficient for hunger and conclude that it is not a key regulator of appetite or weight gain in mice. Ghrelin's only essential role in mice appears to be the maintenance of plasma glucose during periods of starvation.Item Identification and Biochemical Characterization of Ghrelin O-Acyltransferase (GOAT)(2009-06-19) Yang, Jing; Goldstein, Joseph L.Ghrelin is a 28-amino acid, appetite-stimulating hormone secreted by the food-deprived stomach. Ser-3 of ghrelin is acylated with an eight-carbon fatty acid, octanoate, which is critically required for its endocrine actions. However, the octanoylating enzyme had remained elusive for nearly a decade. By expression cloning, I have identified GOAT (Ghrelin O-Acyltransferase), an enzyme belonging to a family of 16 polytopic membrane-bound O-acyltransferases. GOAT activity requires catalytic Asn and His residues, which are conserved through vertebrates. Consistent with its function, GOAT mRNA is largely restricted to stomach and intestine, the major ghrelin-secreting tissues. To further characterize GOAT function biochemically, I have developed a robust in vitro assay using membranes from insect cells infected with baculovirus encoding recombinant mouse GOAT. GOAT-containing membranes catalyze the transfer of [3H]octanoyl from [3H]octanoyl CoA to recombinant proghrelin in vitro. 50 microM palmitoyl CoA is necessary in the assays to prevent the deacylation of [3H]octanoyl CoA by crude membrane preparations. Maximal GOAT activity is observed at pH 7.0, and there is no apparent requirement for metals as determined by a lack of inhibition by 1 mM EDTA. The apparent Km for proghrelin is 6 microM and for [3H]octanoyl CoA is 0.6 microM. The octanoylation reaction strictly depends on the GOAT recognition site comprising three of the four N-terminal amino acids of proghrelin: Gly-1, Ser-3, and Phe-4. A pentapeptide containing only the N-terminal five amino acids of ghrelin is octanoylated by the enzyme. Moreover, I have demonstrated that the activity of GOAT is subjected to end-product inhibition. Together, the insights provided by my research may facilitate the design of useful inhibitors of GOAT.Item Investigating the Enteroendocrine - Brain Axis: Ghrelin Cell and ECL Cell Physiology and Ghrelin Action on Mood and Complex Eating(2014-06-11) Walker, Angela Kay; Eisch, Amelia J.; Powell, Craig M.; Scherer, Philipp; Zigman, Jeffrey M.The mechanisms and neurochemical pathways through which the orexigenic peptide hormone ghrelin act to regulate homeostatic feeding is fairly well documented. However, less understood are the mechanisms and brain regions that mediate ghrelin's effects on mood and complex eating behaviors. At the cellular level, little is known about the ghrelin cell's transcriptional profile, its secretory products other than ghrelin, and its relationship to other gastric endocrine cells, such as the histamine producing enterochromaffin-like cell. My doctoral research encompasses multiple aspects of the ghrelin system, from physiological assessments of the ghrelin cell to evaluations of ghrelin action on cue-potentiated feeding and stress-induced depressive-like behavior. Ghrelin has antidepressant effects, which become obvious following chronic stress. In the first part of my thesis, I found that this effect was mediated by neurogenesis. I observed that chronic stress reduces neurogenesis more severely in the ventral dentate gyrus of Ghsr-null mice, suggesting ghrelin provides a level of neuroprotection in the stress environment. Administration of anti-apoptotic P7C3-related compounds not only blocked stress-induced reductions in neurogenesis, but also minimized the severity of depressive-like behavior in mice. Focal hippocampal irradiation prevented the anti-depressant efficacy of P7C3-related compounds, indicating that P7C3 regulates mood directly through neurogenesis. In the second part of my thesis, I designed a novel protocol for studying cue-potentiated feeding behaviors in mice. Absence of ghrelin signaling in Ghsr-null mice, or administration of a ghrelin receptor antagonist in wild-type mice, disrupted the development of normal cue-food associations. Additionally, I discovered Ghsr expression in the basolateral amygdala (BLA), and BLA neuronal activation in response to a food-associated positive cue significantly correlated with amount of food intake. Thus, ghrelin signaling in the BLA may be responsible for its mediation of cue-potentiated feeding behaviors. The third part of my thesis examined the ghrelin cell transcriptome for potential secretory proteins and revealed significant expression of Rbp4, Ttr, and Nucb2, along with RBP4 protein secretion. Lastly, I characterized a novel HDC-Cre mouse model that may be advantageous in future studies to determine potential interactions between histaminergic and ghrelin signaling pathways. The full range of these discoveries advances our comprehensive understanding of ghrelin.Item Role of Arcuate AgRP Neurons in Ghrelin Action on Food Reward(2014-02-04) Mosher, Christina; Wang, Qian; Osborne-Lawrence, Sherri; Zigman, JeffreyMedia coverage of the current obesity epidemic often ascribes stress as a major cause of excessive food intake in humans, with a particular increase in calorically dense 'comfort foods.' In the current study, the hormonal and neural link between stress and feeding behavior was further investigated using behavioral tests to monitor the rewarding effects of high fat diet in a novel transgenic mouse model. In particular, the octanoylated peptide hormone, ghrelin, plays a key role in appetite regulation as well as a behavioral role in the stress response. Synthesized and secreted by a small population of gastric epithelial cells, ghrelin is the only circulating hormone known to stimulate appetite. Importantly, ghrelin levels are significantly and persistently elevated in response to chronic stress. Also, ghrelin signaling enables the development of stress-induced conditioned place preference (CPP) for high fat diet, a reward-based eating behavior, and ghrelin minimizes stress-induced depressive-like behavior. The ghrelin receptor, known as the growth hormone secretagogue receptor (GHSR), is highly expressed in hypothalamic brain regions involved in homeostatic feeding, such as the arcuate nucleus (Arc). Preliminary studies in the lab indicate that acute ghrelin-induced feeding, which is not observed in GHSR-deficient (GHSR-null) mice, is partially restored in mice with GHSR expression only in Arc AgRP neurons. We now hypothesize that the GHSRs present on Arc AgRP neurons are sufficient to mediate the stress-induced development of food reward behavior in mice. To test this hypothesis, the lab has generated a novel, genetically engineered mouse line in which GHSR expression is limited only to Arc AgRP neurons. Such is possible due to a tamoxifen-inducible Cre recombinase, which in turn removes a loxP-flanked transcriptional blocking cassette from an altered GHSR gene, thus allowing tamoxifen-dependent, AgRP neuron-selective reactivation of GHSR expression. For these studies, littermates of 4 genotypes were used: wild-type mice, wild-type mice expressing Cre recombinase, GHSR-null mice, and mice with GHSRs expressed only in AgRP neurons, as described above. All mice were treated with tamoxifen for five days to activate Cre recombinase, allowed to recover for three weeks and monitored for ad-lib food intake for the last nine days of recovery. Mice were then subjected to a ten-day chronic social defeat stress (CSDS) protocol and afterward tested for depressive-like behavior using the social interaction (SI) test. Following the SI test, mice underwent a CPP for HFD protocol, which includes a pretest, twelve days of conditioning, and a test day. Data for an initial cohort of animals has been obtained and analyzed, and studies with additional cohorts are ongoing. The new mouse model with GHSRs expressed only in the Arc will assist in determining whether AgRP neurons in the Arc are sufficient to mediate ghrelin's effects on stress-induced food reward-behavior and stress-associated depressive-like behavior. Further work may include a similar tamoxifen-inducible, Cre-recombinase system expressing GHSRs only in the VTA or only in the hippocampus to narrow down where ghrelin has its food reward behavior effects.Item The role of ghrelin in body weight regulation, reward behavior and mood(2008-12-12) Zigman, Jeffrey M.Item [UT Southwestern Medical Center News](2008-06-15) Shear, Kristen HollandItem [UT Southwestern Medical Center News](2011-06-27) Shear, Kristen HollandItem [UT Southwestern Medical Center News](2009-12-28) McKenzie, Aline