Metabolism
Oral Presentations
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The Effects of Maternal Obesity and Consumption of a High-fat Diet on the Development of Fetal Hepatic Inflammation in the Non-human Primate.
Wilmon Grant, Wilmon F. Grant, Melanie B. Gillingham, Theodore P. Braun, Peter R. Levasseur, Xin Xia Zhu, Sarah M. Williams, Kevin L. Grove, Daniel L. MarksKeywords: Non-Alcoholic Fatty Liver Disease, High Fat Diet, Metabolic Programming Abstract:Childhood obesity is occurring in epidemic proportions. While associations between maternal obesity and diet have been suggested, their contributions to metabolic programming in the offspring are not well understood. We have developed a unique non-human primate model to address the impact that maternal obesity and nutrient excess has on the developing fetus. Age and weight matched female Japanese macaques were divided into two diet groups; Control (13% fat) or High Fat (35% fat with high levels of saturated, monounsaturated and ω-6 fatty acids). Each year pregnancies were terminated during the early 3rd trimester and fetal blood and tissue were collected for analysis. In the high fat diet group, we found that fetal livers had significant upregulation of many pro-inflammatory genes including: interleukin-13, lymphotoxin-α, interleukin-1β and tumor necrosis factor-α. Protein analysis showed nearly two-fold increases in interleukin-13 and lymphotoxin-α protein levels in these livers. In addition, the ω-6/ω-3 fatty acid ratios in both maternal and fetal plasma were significantly increased in the high-fat diet group. Importantly, while ω-3 derived metabolites are anti-inflammatory and anti-thrombotic, ω-6 metabolites are proinflammatory and prothrombotic. Our results suggest that that a high circulating ω-6/ω-3 ratio may shift both mother and fetus to a pro-inflammatory physiologic state. In addition, the upregulation of hepatic markers involved in inflammation, fibrosis and atherosclerosis suggest that non-alcoholic fatty liver disease is present and permanent damage may be occurring in these fetal livers.
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Breastfeeding Success Among Infants with PKU
Sandra Banta Wright, Kathleen Shelton PhD PNP, Kathleen Knafl PhD FAAN, Nancy Lowe PhD CNM FAANKeywords: Breastfeeding, Pku Abstract:Problem
•There is a paucity of research about breastfeeding for infants with PKU & few research-based guidelines for clinical care.
Aim
To compare phenylalanine (Phe) levels between breast-fed and formula-fed infants with PKU
Sample
Infant selection criteria:
•Diagnosed with PKU between January 1, 1980 and December 31, 2005
•Received medical and dietary management of PKU by one month of age
Method
•Retrospective chart review
•Investigator developed data collection sheet
Results
•The data indicate that the breastfeeding incidence continues to increase at the time of diagnosis.
•During the first year, the majority of infants (breast-fed or formula-fed) have similar mean Phe values (66% vs 68%).
Chi square analysis revealed: χ2 (df = 2, N = 3,260) = 205, p 6mg% (37% vs 37%).
These results are remarkable in that mothers who were managing PKU , a complex disorder, were able to also breastfeed their infant successfully.
Implications for Further Research •To explore how mothers successfully maintain breastfeeding while managing the PKU disorder for their infants. •To identify effective strategies for the clinical management of breastfeeding & PKU .
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Two KATP Channel Mutants with Congenital Hyperinsulinism Disease Phenotype Exhibit Diabetes-Inducing Gating Properties
Emily Pratt, FF Yan, YW Lin, CA Stanley, SL ShyngKeywords: Potassium Channel, Diabetes Abstract:ATP -sensitive potassium (KATP) channels mediate glucose-induced insulin secretion by coupling metabolic signals to beta-cell membrane potential. Reduced KATP channel expression or loss of channel response to Mg•ADP lead to poor channel function, resulting in congenital hyperinsulinism (CHI). Recently, we found that two mutations in the SUR1 gene, R74W and E128K identified from CHI patients, cause reduced KATP channel surface expression and that this defect was able to be rescued by sulfonylureas (SU), such as glibenclimide or tolbutamide. The same phenomenon was observed in an insulin-secreting cell line (INS-1 cell) and primary rat islets expressing the mutants. Interestingly, after rescue to the cell surface and wash-out of the SU chaperone, E128K and R74W mutant channels show reduced ATP sensitivity, a gating defect commonly associated with permanent neonatal diabetes. This is expected lead to inhibition of insulin secretion, suggesting an expression level-dependent reversal from hyperinsulinism to a diabetes phenotype for the R74W and E128K KATP channels. Consistent with this hypothesis, electrophysiological recordings of E128K and R74W expressing INS -1 cells showed hyperpolarized membrane potentials following SU rescue and 12mM glucose stimulation. Additionally, single channel properties of the two mutant channels indicate they have decreased open-probability, suggestive of reduced coupling between the SUR1 and Kir6.2 subunits. Our findings provide a novel disease paradigm in which the expression level of the mutant dictates presentation of two opposing disease phenotypes.
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AAV8-mediated correction of a metabolic renal disease via gene repair in vivo.
Nicole Paulk, Karsten Wursthorn, Milton Finegold, Mark Kay, Markus GrompeKeywords: Mouse Model, Renal Metabolic Disease, Gene Correction, Fah, Aav Abstract:Renal gene therapy has proven challenging despite successful advances in gene delivery to other organ systems. Some reports have shown that rAAV serotypes (1, 2 and 5) have renal tropism and are capable of stable transgene expression in renal cells in vitro. However, rAAV-based gene correction in a renal disease model in vivo has not been reported.
Our previous work has shown rAAV serotype 8 capable of in vivo correction of the metabolic liver disease hereditary tyrosinemia type 1 (HT1). HT1 stems from lack or malfunction of fumarylacetoacetate hydrolase (Fah), the final enzyme in the tyrosine catabolic pathway. Fah deficiency also affects renal proximal tubule function, creating a renal Fanconi syndrome. Thus, we expanded our analysis to attempt functional correction of renal HT1 .
Our vector carries 4.5 Kb of partial genomic sequence from the mouse Fah locus. Importantly, Fah expression can be restored only by chromosomal integration by homologous recombination. The rAAV8 viral vector was given by tail vein injection into Fah5961SB mice, a well described point mutation-based model for HT1 . After 3 months selection, greater than 50% of renal proximal tubules were corrected as shown by Fah immunohistochemistry. Southern blot and urinalysis will be completed to demonstrate correction of treated mice when compared to control wild-type mice.
The results demonstrate that rAAV8 is capable of mediating stable targeted gene correction of renal proximal tubules in vivo. Moreover, rAAV8 has a previously undescribed renal tropism.
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Cross-talk between metal binding sites regulates ATP7B activity
Erik Le Shane, Barry AN, Walker JM, Blackburn N, Shinde U, Lutsenko SKeywords: Wilson'S Disease, Atp7b, Copper Abstract:The N-Terminal domain of copper-transporting ATPase ATP7B (N-ATP7B) is involved in regulating the protein’s catalytic activity and intracellular trafficking. The domain contains at least six metal sites, each of which binds one copper molecule within the conserved GMTCXXC motif. N-ATP7B is also a site of copper delivery by the metallochaperone Atox1. We have previously shown that in the full-length N-ATP7B, the metal binding site 2 (MBS2) is the preferred site for initial transfer of copper by Atox1. The CxxC>AxxA mutation in MBS2 impairs the catalytic activation of ATP7B by Atox1, but not by free copper. We now demonstrate that this mutation also reduces the metal binding capability of other metal binding sites in N-ATP7B, indicative of cross-talk between the N-terminal metal binding sites. Our results suggests that MBS2 and MBS3 , but not MBS6 , are involved in interdomain interactions that are essential for correct copper-binding stoichiometry of the entire N- ATP7B . The effect of mutations on overall conformation of N- ATP7B was confirmed by limited proteolysis that revealed distinct proteolytic patterns for the control and mutant N- ATP7Bs. We also demonstrate that the MBS2 and MBS3 mutants are prone to rapid cysteine oxidation, but maintain the same copper coordination. In silica modeling suggests that MBS2 may be essential as a gateway for copper and electron transfer due to the preferential exposure of the metal binding loop, which is more likely to be in an open conformation than that of other metal binding sites.