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Biomedical subjects

Keith E Georgeson

Publications and source records attributed to Keith E Georgeson.

15 recordsLinked to original sources
PubMed2006

Mechanism of oleoylethanolamide on fatty acid uptake in small intestine after food intake and body weight reduction.

The increase in the prevalence of human obesity highlights the need to identify molecular and cellular mechanisms involved in control of feeding and energy balance. Oleoylethanolamide (OEA), an endogenous lipid produced primarily in the small intestine, has been identified to play an important role in the regulation of animal food intake and body weight. Previous studies indicated that OEA activates peroxisome proliferator-activated receptor-alpha, which is required to mediate the effects of appetite suppression, reduces blood lipid levels, and enhances peripheral fatty acid catabolism. However, the effect of OEA on enterocyte function is unclear. In this study, we have examined the effect of OEA on intestinal fatty acid uptake and FAT/CD36 expression in vivo and in vitro. We intraperitoneally administered OEA to rats and examined FAT/CD36 mRNA level and fatty acid uptake in enterocytes isolated from the proximal small intestine, as well as in adipocytes. Our results indicate that OEA treatment significantly increased FAT/CD36 mRNA expression in intestinal mucosa and isolated jejunal enterocytes. In addition, we also found that OEA treatment significantly increases fatty acid uptake in isolated enterocytes in vitro. These results suggest that in addition to appetite regulation, OEA may regulate body weight by altered peripheral lipid metabolism, including increased lipolysis in adipocytes and enhanced fatty acid uptake in enterocytes, both in conjunction with increased expression of FAT/CD36. This study may have important implications in understanding the mechanism of OEA in the regulation of fatty acid absorption in human physiological and pathophysiological conditions.

Adipocytes↗
PubMed2006

The role of hyperglycemia in FAT/CD36 expression and function.

FAT/CD36 is a long-chain fatty acid transporter and scavenger receptor for oxidized LDL. Defects in FAT/CD36 have been linked to the hypertriglyceridemia and insulin resistance. Expression of FAT/CD36 was reported increase in type 1 diabetes; however, it remains unclear whether serum glucose or insulin plays an important role in this regulation. To elucidate the individual contribution of plasma glucose and insulin in the regulation of FAT/CD36 mRNA expression, we induced type 1 diabetes in male Sprague-Dawley rats using streptozotocin (STZ) and compared traditional insulin treatment with administration of the orally absorbed chemical agent vanadate, which reduces blood glucose levels via mechanisms that bypass insulin receptor action. STZ-exposed animals showed significant decreases in body weight (285.5 +/- 2.8 vs. 233.1 +/- 3.5 g, P < 0.001) and serum insulin levels (9.7 +/- 0.7 vs. 2.8 +/- 0.6 microU/ml, P < 0.05), accompanied by significant increases in blood glucose (71 +/- 3 vs. 433 +/- 11 mg/dl, P < 0.001), water intake (38.9 +/- 0.9 vs. 205.9 +/- 3.3 ml/day, P < 0.001) and food intake (22.0 +/- 0.4 vs. 36.9 +/- 1.0 g/day, P < 0.001). Diabetic animals demonstrated significant increases in FAT/CD36 mRNA levels in duodenum (2.2-fold), jejunum (1.8-fold), ileum (1.5-fold), adipose tissue (1.7-fold), and heart (2.5-fold) (P < 0.05). Insulin treatment reversed body weight loss and corrected hyperglycemia at diabetic rats as expected. Insulin treatment also corrected increased FAT/CD36 mRNA expression at diabetic rats. Vanadate significantly reduced serum glucose levels without increasing serum insulin or affecting body weight but reversed increased FAT/CD36 mRNA expression in diabetic rats. These data suggest that plasma glucose levels play more important role in the regulation of FAT/CD36 expression than concurrent changes in plasma insulin.

Adipose Tissue↗
PubMed2006

Molecular basis of melanocortin-4 receptor for AGRP inverse agonism.

We have investigated receptor structural components of the melanocortin-4 receptor (MC4R) responsible for ligand-dependent inverse agonism. We utilized agouti-related protein (AGRP), an inverse agonist which reduces MC4R basal cAMP production, as a tool to determine the molecular mechanism. We tested a series of chimeric receptors and utilized MC4R and MC1R as templates, in which AGRP is an inverse agonist for MC4R but not for MC1R. Our results indicate that replacements of the extracellular loops 1, 2 and 3 of MC4R with the corresponding regions of MC1R did not affect AGRP inverse agonist activity. However, replacement of the N terminus of MC4R with the same region of MC1R decreases AGRP inverse agonism. Replacement of transmembrane domains 3, 4, 5 and 6 of MC4R with the corresponding regions of MC1R did not affect AGRP inverse agonist activity but mutation of D90A in transmembrane 2 (TM2) and D298A in TM7 abolished AGRP inverse activity. Deletion of the distal MC4R C terminus fails to maintain AGRP mediated reduction in basal cAMP production although it maintains NDP-MSH mediated cAMP production. In conclusion, our results indicate that the N terminus and the distal C terminus of MC4R do appear to play important roles in AGRP inverse agonism but not NDP-MSH mediated receptor activation. Our results also indicate that the residues D90 in TM2 and D298 in TM7 of hMC4R are involved in not only NDP-MSH mediated receptor activation but also AGRP mediated inverse agonism.

Agouti-Related Protein↗
PubMed2006

Functional characterization of the modified melanocortin peptides responsible for ligand selectivity at the human melanocortin receptors.

The melanocortin system plays an important role in energy homeostasis as well as skin pigmentation, steroidogenesis and exocrine gland function. In this study, we examined eight Ac-His-Phe-Arg-Trp-NH(2) tetrapeptides that were modified at the Phe position and pharmacologically characterized their activities at the human MCR wild-types and their mutants. Our results indicate that at the hMC1R, all D stereochemical modified residues at the Phe position of peptides increase cAMP production in a dose-dependent manner. At the hMC3R, the DPhe peptide dose dependently increases cAMP production but all other three tetrapeptides were not. At the hMC4R, both the DPhe and DNal(1') peptides induce cAMP production. However, both DTyr and DNal(2') were not able to induce cAMP production. Further studies indicated that at the hMC1R M128L mutant receptor, the all D-configured tetrapeptides reduce their potencies as compared to that of hMC1R wild-type. However, at the hMC3R and hMC4R L165M and L133M mutant receptors, the DNal(2') and DTyr tetrapeptides possess agonist activity. These findings indicate that DPhe in tetrapeptide plays an important role in ligand selectivity and specific residue TM3 of the melanocortin receptors is crucial for ligand selectivity.

Amino Acid Sequence↗
PubMed2006

Molecular characterization of human melanocortin-3 receptor ligand-receptor interaction.

UNLABELLED: Melanocortin-3 receptor (MC3R), primarily expressed in the hypothalamus, plays an important role in the regulation of energy homeostasis. MC3R-deficient (MC3R(-)(/)(-)) mice demonstrate increased fat mass, higher feeding efficiency, hyperleptinaemia, and mild hyperinsulinism. At least one specific mutation of MC3R has been identified to be associated with human obesity. Functional analysis of this altered MC3R (I183N) has indicated that the mutation completely abolishes agonist-mediated receptor activation. However, the specific molecular determinants of MC3R responsible for ligand binding and receptor signaling are currently unknown. The present study is to determine the structural aspects of MC3R responsible for ligand binding and receptor signaling. On the basis of our theoretical model for MC1R, using mutagenesis, we have examined 19 transmembrane domain amino acids selected for these potential roles in ligand binding and receptor signaling. Our results indicate that (i) substitutions of charged amino acid residues E131 in transmembrane domain 2 (TM2), D154 and D158 in TM3, and H298 in TM6 with alanine dramatically reduced NDP-MSH binding affinity and receptor signaling, (ii) substitutions of aromatic amino acids F295 and F296 in TM6 with alanine also significantly decreased NDP-MSH binding and receptor activity, (iii) substitutions of D121in TM2 and D332 in TM7 with alanine resulted in the complete loss of ligand binding, ligand induced receptor activation, and cell surface protein expression, and (iv) interestingly, substitution of L165 in TM3 with methionine or alanine switched antagonist SHU9119 into a receptor agonist. IN CONCLUSION: Our results suggest that TM3 and TM6 are important for NDP-MSH binding, while D121 in TM2 and D332 in TM7 are crucial for receptor activity and signaling. Importantly, L165 in TM3 is critical for agonist or antagonist selectivity. These results provide important information about the molecular determinants of hMC3R responsible for ligand binding and receptor signaling.

Amino Acid Sequence↗
PubMed2006

Revision Nissen fundoplication can be completed laparoscopically with a low rate of complications: a single-institution experience with 72 children.

PURPOSE: Recurrent gastroesophageal reflux is a common complication after fundoplication and is often treated with revision fundoplication. We report our experience with laparoscopic redo fundoplication. METHODS: The medical records of all patients in whom laparoscopic revision fundoplication was attempted over a 7 1/2-year period were reviewed. RESULTS: Redo laparoscopic fundoplication was attempted in 72 pediatric patients. Ten patients had undergone initial open fundoplication, and 9 additional patients had prior abdominal surgery. Fifty-one percent of patients were neurologically impaired. Laparoscopic fundoplication was completed in 89% of first-time redo operations and 68% of second revisions with average operative times of 2.2 +/- 1.0 and 2.6 +/- 0.9 hours, respectively. Herniation of the fundoplication through the hiatus was common (75%) and the fundoplication was intact in 49%. Conversions to laparotomy were because of difficulties with dissection or visualization. No patients required intraoperative transfusion. No patients required reoperation in the perioperative period. There were no perioperative deaths. Twenty-six percent of the 72 patients went on to a third operation for gastroesophageal reflux, and 4 of these had a fourth. CONCLUSION: Revision laparoscopic fundoplication is a technically challenging operation but can usually be completed and is characterized by a low rate of complications.

Adolescent↗
PubMed2005

Molecular mechanism of the intracellular segments of the melanocortin-4 receptor for NDP-MSH signaling.

Mutations of the human melanocortin-4 receptor (hMC4R) have been previously identified to be the most common cause of monogenic human obesity. Specifically, mutations of the intracellular C terminus and the third intracellular loop of hMC4R have been reported to play an important role in human obesity. However, the molecular basis of these hMC4R intracellular segments in receptor function remains unclear. In this study, we utilized deletions and mutations of specific portions of the hMC4R to determine the molecular mechanism of both the C terminus and the third intracellular loop in receptor signaling. Our results indicate that deletions of the distal 25 (the entire C terminus), 22, 18, 17, 16, and 15 amino acids of the C terminus result in the complete loss of both [Nle(4)-d-Phe(7)]-alpha-melanocyte stimulating hormone (NDP-MSH) binding and NDP-MSH-mediated cAMP production. Deletion of the distal 14 amino acids of the C terminus significantly decreases both NDP-MSH binding affinity and potency, but deletion of the distal 13 amino acids of the C terminus does not affect NDP-MSH activity. Further analysis revealed that the proximal 12 amino acids of the C terminus are not only important for receptor signaling but also important for ligand binding. Our results also indicate that the third intracellular loop of the hMC4R is important for receptor signaling but not ligand binding. In summary, our findings suggest that the proximal region of the melanocortin-4 receptor (MC4R) C terminus is crucial not only for receptor signaling but also for ligand binding, while the third intracellular loop is important mainly for receptor signaling.

Amino Acid Sequence↗
PubMed2004

Laparoscopic-assisted approaches for the definitive surgery for Hirschsprung's disease.

The surgical management of Hirschsprung's disease has progressed from a two- or three-stage procedure to a primary operation over the last 25 years. More recently, definitive surgery for Hirschsprung's disease has been performed using minimally invasive techniques. The Swenson, Duhamel and Soave endorectal pull-through procedures have all been reported using minimally invasive approaches. The endorectal dissection has become the dominant minimal access procedure because of the ease and reliability in performing this technique and the excellent results obtained. Although a transanal endorectal pull-through can be performed without laparoscopy, the laparoscopic-assisted transanal endorectal pull-through is a much more versatile technique and allows early biopsies to determine the extent of aganglionic and dysfunctional bowel before ablation of the rectum and mesocolon. The authors use a laparoscopic-assisted transanal pull-through for aganglionosis of the left and transverse colon. Total colon aganglionosis or aganglionosis of the ascending colon is managed by a laparoscopic-assisted Duhamel procedure which provides a better reservoir in patients with a short or absent colon.

Child, Preschool↗
PubMed2004

Minimally invasive surgery in the neonate: review of current evidence.

Minimally invasive surgery has been one of the most important surgical advances in the last 15 years. The development of smaller instruments has allowed pediatric surgeons to apply this rapidly evolving technology to neonates. Congenital neonatal deformities including tracheoesophageal fistula, patent ductus arteriosus, duodenal atresia and anorectal malformations are now being managed with minimally invasive surgery. This article summarizes the status of these techniques in neonates.

Bronchopulmonary Sequestration↗
PubMed2003

Molecular determination of agouti-related protein binding to human melanocortin-4 receptor.

Agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin-4 receptor (MC4R) that functions in the hypothalamic control of feeding behavior. Our previous studies have suggested that in addition to exoloops 2 and 3, several transmembrane domains of MC4R may be important for AGRP binding. However, the detailed molecular basis of MC4R domains in AGRP binding is presently unclear. The present studies were designed to determine the specific contribution of MC4R exoloops and transmembrane domains to AGRP binding by using chimeric receptor constructs of the human melanocortin-1 receptor (hMC1R), a receptor that is not inhibited by AGRP, and the human MC4R (hMC4R), a receptor that is potently inhibited by AGRP. Our results indicate that substitutions of the second and third extracellular loops of the MC4R with homologous domains of the MC1R dramatically decreased AGRP 87-132 binding affinity, but did not affect AGRP 110-117 binding affinity. In contrast, cassette substitutions of the third or fourth transmembrane domain of the MC4R with the homologous domain of the MC1R resulted in a substantial decrease of AGRP 87-132 binding affinity and loss of AGRP 110-117 binding affinity. These data suggest that the AGRP fragment 110-117 has no binding sites at exoloops of hMC4R and that transmembrane domains of MC4R may play an important role in AGRP 110-117 binding and function, whereas the exoloops do not. The second and third extracellular loops of MC4R are important for AGRP 87-132 N-terminal binding, whereas the third and fourth transmembrane domains of hMC4R are crucial for AGRP 110-117 binding.

Agouti-Related Protein↗
PubMed2002

Molecular determinants of human melanocortin-4 receptor responsible for antagonist SHU9119 selective activity.

The hypothalamic melanocortin-4 receptor (MC4R), a seven transmembrane G-protein-coupled receptor, plays an important role in the regulation of body weight. The synthetic melanocortin analog SHU9119 has been widely used to characterize the physiological role of MC4R in feeding behavior and energy homeostasis. Previous studies indicated that SHU9119 is an agonist at the melanocortin-1 receptor (MC1R) but an antagonist at the MC4R. However, the molecular basis of the interaction between hMC4R and SHU9119 has not been clearly defined. To gain insight into the molecular determinants of hMC4R in the selectivity of SHU9119 chimeras and mutants hMC1R and hMC4R were expressed in cell lines and pharmacologically analyzed. A region of receptor containing the third transmembrane of hMC4R was found to be required for selective SHU9119 antagonism. Further mutagenesis studies of this region of hMC4R demonstrated that the amino acid residue leucine 133 in the third transmembrane was critical for the selective antagonist activity of SHU9119. The single substitution of leucine 133 to methionine did not affect SHU9119 binding to hMC4R. However, this substitution did convert SHU9119 from an antagonist to an agonist. Conversely, exchange of Met(128) in hMC1R to Leu, the homologous residue 133 of hMC4R, displayed a reduction in SHU9119 binding affinity and potency. This report provides the details of the molecular recognition of SHU9119 antagonism at hMC4R and shows that amino acid Leu(133) of hMC4R plays a key role in melanocortin receptor subtype specificity.

Amino Acid Sequence↗
PubMed2002

Laparoscopic-assisted pull-through for Hirschsprung's disease.

There appears to be a trend in the management of Hirschsprung's disease toward a one-stage surgical correction in the newborn period. After the development of the one-stage laparoscopic-assisted endorectal pull-through in 1995, the minimally invasive approaches have come to dominate the surgical management of this disease. The laparoscopic-assisted pull-through is ideally suited for those patients with aganglionosis confined to the rectum, sigmoid, or proximal left colon. At the time of the operation, a laparoscopic biopsy is recommended to identify, with certainty, the site of the transition zone before performing the colonic mobilization. Advantages of this approach include reduced discomfort, the early initiation of defecation through a normally positioned ganglionic colon and rectum, and reduced postoperative hospitalization.

Child↗
PubMed2002

Laparoscopic-assisted total colectomy with pouch reconstruction.

Whether in adults or children, benign diseases of the colon are ideally suited for laparoscopic-assisted resection. In children, the most frequent diseases leading to colectomy and pull-through include Hirschsprung's disease, ulcerative colitis, and familial polyposis. Laparoscopic proctocolectomy with J-pouch reconstruction is an ideal approach for children and adolescents who are generally stable but suffer from unremitting symptoms of ulcerative colitis. An early experience with this technique at the Children's Hospital in Alabama is described for children with ulcerative colitis.

Adolescent↗
PubMed2002

Importance of Sp1 consensus motifs in the MYCN promoter.

BACKGROUND: MYCN (N-myc) amplification in neuroblastoma is associated with poor clinical outcome. Factors that regulate MYCN expression have not been elucidated. MYCN is considered a TATA-less promoter, whereas significant promoter activity resides within 160 bp 5' of the major transcription start site. This region contains two GC-rich motifs and a CT box, regions for potential transcription factor interaction. METHODS: To characterize DNA-protein interactions in this region of the MYCN promoter, electrophoretic mobility shift assays, and promoter-reporter were used. RESULTS: A MYCN promoter fragment was incubated with HeLa nuclear extract, with or without competitors. Three major protein/DNA complexes were formed. Formation of 2 complexes could be inhibited by unlabeled Sp1 consensus duplex and by the Sp1 site-specific drug WP631. Purified Sp1 protein produced a complex similar to that formed with HeLa extract. To determine whether these DNA/protein interactions could be blocked in a sequence-specific fashion, a triplex forming oligonucleotide (TFO) was used. This TFO was designed to bind in the major groove of the promoter, covering the CT-box (putative Sp1 binding) motif. When triplex formation was followed by addition of nuclear extract, protein binding was indeed inhibited. Functional significance of this inhibition was tested with pE/Bnmyc-luc, a promoter-reporter plasmid containing the human MYCN promoter driving luciferase expression. Incubation with TFO, but not control oligodeoxynucleotides, completely inhibited luciferase activity. CONCLUSIONS: These data suggest that protein binding does occur in regions of the MYCN promoter containing GC and CT box elements and that this interaction is important for MYCN promoter activity. By inference, these data also suggest that the proteins that bind in this region are Sp1 family members.

Base Sequence↗