Browsing by Subject "Acyltransferases"
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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 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 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 Mechanisms of Genome Buffering and Cell Fate Coordination in Adult Tissue Homeostasis(2016-07-26) Tuladhar, Rubina; Amatruda, James F.; Scherer, Philipp; DeBerardinis, Ralph J.; Lum, LawrenceSelf-renewal competency of adult stem cells is essential for tissue homeostasis. The corruption of genes essential for genome preservation or for niche-stem cell interactions frequently results in loss of stem cell viability and disease. The two components of my thesis focus on understanding adult stem cell preservation - the integration of metabolism and intercellular communication mediated by the Wnt family of secreted signaling molecules, and epigenetic mechanisms that buffer the proteome against insertion/deletion (INDEL)-type genetic mutations. Wnt-mediated signaling is essential for embryogenesis and the maintenance of adult tissues. Lipidation of Wnt proteins by the acyltransferase Porcupine (Porcn) is crucial for secretory pathway exiting. Using chemically based approaches, I have demonstrated that Porcn active site features conserved across animals enforce ω-7 cis fatty acylation of Wnt proteins. Deviant acylation of a Wnt protein using an exogenously supplied trans fatty acid cripples its ability to traverse the secretory pathway due to a previously unappreciated stereoselectivity of the Wnt chaperone Wntless (WLS) for fatty acids. My findings provide a mechanistic account of chemical specificity observed in Porcn inhibitors, and delineate a universal mechanism for integrating communal cell fate decision-making with metabolic fitness. As part of my efforts to generate isogenic cells for the expression of LKB1, a tumor suppressor that regulates Wnt protein production, I encountered the emergence of foreign LKB1 proteins subsequent to the introduction of INDELs by the DNA editing enzyme CRISPR-Cas9. I demonstrate that these novel proteins are the products of: a) the installation of internal ribosomal entry sites (IRES), b) the induction of exon skipping due to compromised exon splicing enhancers (ESEs), and c) the conversion of pseudo-mRNAs to protein-coding mRNAs due to the unwanted elimination of premature termination codons. I propose that these molecular events serve as compensatory mechanisms employed by cells to restore proteome integrity in the face of INDEL-type challenges to the genome posed by pathogens and environmental mutagens. Taken together, these two projects will: a) delineate intervention strategies premised upon the attack of an universally conserved point of intersection between metabolism and cell-to-cell communication, b) facilitate the personalization of medicine, and c) accelerate tissue engineering initiatives.