<p>This thesis examines the effect of surgical weight loss on gut and adipose tissue peptides involved in appetite regulation and energy homeostasis in morbidly obese humans. Roux-en-Y gastric bypass (RYGBP) is the gold standard operation used for effective long-term weight loss and improved health. The exact mechanisms for this outcome are under investigation.</p><p>We measured ghrelin, a recently discovered hunger hormone, insulin, adiponectin and leptin along with anthropometry measures in 66 morbidly obese patients prior to and 6 and 12 months after RYGBP. Impressive weight loss occurred postoperatively as did alterations in the peptides. Consistent correlations were found between weight, leptin, ghrelin and insulin. The main findings were low ghrelin concentrations in obesity and an increase after RYGBP.</p><p>We explored inflammatory proteins C-reactive protein (CRP), serum amyloid A and interleukin-6 before and during massive weight loss 6 and 12 months after RYGBP in morbidly obese subjects. The studied proteins declined after surgery and a correlation between CRP and homeostatic model of assessment for insulin resistance, independent of BMI, strongly linked insulin resistance and inflammation. CRP declined most in insulin-sensitive subjects.</p><p>We examined the excluded stomach mucosa and vagus nerve by measuring gastrin, pepsinogen I (PGI), pancreatic polypeptide (PP) and ghrelin levels during week 1 and year after RYGBP. Ghrelin levels rose with weight loss but declined 24-hours after surgery, like PP, indicating transient vagal nerve damage. Low levels of gastrin and PGI suggest a resting mucosa.</p><p>We evaluated gut peptides: peptide YY (PYY), glucaogon like peptide-1 (GLP-1), pro-neurotensin (pro-NT) and PP, in lean (young and middle-aged), obese and postoperative RYGBP subjects pre- and postprandially. RYGBP subjects had exaggerated levels of PYY and GLP-1 postprandially and higher basal proNT levels, implying a ‘satiety peptide tone’ that may contribute to the maintenance of weight loss.</p><p>In summary, RYGBP results in marked weight loss and alterations in gut and adipose tissue peptides involved in appetite regulation and energy homeostasis. These postoperative peptide changes may contribute to impressive weight loss observed after RYGBP.</p>

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This thesis examines the effect of surgical weight loss on gut and adipose tissue peptides involved in appetite regulation and energy homeostasis in morbidly obese humans. Roux-en-Y gastric bypass (RYGBP) is the gold standard operation used for effective long-term weight loss and improved health. The exact mechanisms for this outcome are under investigation. We measured ghrelin, a recently discovered hunger hormone, insulin, adiponectin and leptin along with anthropometry measures in 66 morbidly obese patients prior to and 6 and 12 months after RYGBP. Impressive weight loss occurred postoperatively as did alterations in the peptides. Consistent correlations were found between weight, leptin, ghrelin and insulin. The main findings were low ghrelin concentrations in obesity and an increase after RYGBP. We explored inflammatory proteins C-reactive protein (CRP), serum amyloid A and interleukin-6 before and during massive weight loss 6 and 12 months after RYGBP in morbidly obese subjects. The studied proteins declined after surgery and a correlation between CRP and homeostatic model of assessment for insulin resistance, independent of BMI, strongly linked insulin resistance and inflammation. CRP declined most in insulin-sensitive subjects. We examined the excluded stomach mucosa and vagus nerve by measuring gastrin, pepsinogen I (PGI), pancreatic polypeptide (PP) and ghrelin levels during week 1 and year after RYGBP. Ghrelin levels rose with weight loss but declined 24-hours after surgery, like PP, indicating transient vagal nerve damage. Low levels of gastrin and PGI suggest a resting mucosa. We evaluated gut peptides: peptide YY (PYY), glucaogon like peptide-1 (GLP-1), pro-neurotensin (pro-NT) and PP, in lean (young and middle-aged), obese and postoperative RYGBP subjects pre- and postprandially. RYGBP subjects had exaggerated levels of PYY and GLP-1 postprandially and higher basal proNT levels, implying a ‘satiety peptide tone’ that may contribute to the maintenance of weight loss. In summary, RYGBP results in marked weight loss and alterations in gut and adipose tissue peptides involved in appetite regulation and energy homeostasis. These postoperative peptide changes may contribute to impressive weight loss observed after RYGBP.

The objective of this thesis was to relate and link the physiological responses of the cells to the electrical responses or output obtained from the TSM acoustic wave sensor. In particular, the device was applied to the study of immortalized murine hypothalamic neurons (mHypoE-38 and -46 cell models) under a variety of conditions and stimuli. Cellular studies which lead to the production of detectable neuronal responses include neuronal deposition, adhesion and proliferation, alteration in the extent of specific cell-surface interactions, actin filament and microtubule cytoskeletal disruptions, effects of cell depolarization, solution tonicity, inhibition of the Na+-K+ pump via ouabain, effects of neuronal synchronization and the effects ligand-receptor interaction (glucagon). In addition, the introduction of drugs, neurotrophic factors (forskolin and beterferon), toxicity agents (NaOH, EtOH) and TiO2 nanoparticles were similarly investigated. A preliminary study conducted with mouse embryonic stem cells showed that not all cell lines are suitable for investigation with the TSM sensor at the current stage of sensor development. It has been found that control studies conducted with water as the solvent and the bare sensor substrate is insufficient to model the behavior of the sensor in the absence of cells. When biological buffers are used in addition to protein coatings the sensor responses are altered in magnitude and direction. To analyze the full range of cellular changes observed on the TSM sensor, the full impedance spectrum is required. As such in this thesis, the series and parallel resonant frequencies, the motional resistance, the maximum phase of the impedance and the static capacitance (fs, fp, Rm, θmax and Co were used to characterize the cellular responses observed. In the presence of cells fs shifts are largely influenced by the damping of the TSM resonator. The formation of cell-surface interactions and hence the increase in coupling and acoustic energy dissipation can be modeled as an additional resistor in the BVD model. Further sensor and cellular changes can be obtained by negating the effects of damping from fs with the use of Rm and θmax.

G-protein coupled receptors (GPCRs) have been shown to act as part of GPCR associated protein complexes (GAPCs) which are required to appropriately transduce downstream signaling pathways leading to specific cellular actions. I hypothesize that there are distinct molecular effectors that couple to the glucagon superfamily of B-class GPCRs (glucagon, GLP-1, GLP-2, GIP receptors) to effect the myriad of reported actions in numerous target cells including regulation of insulin secretion, intestinal growth and appetite suppression. GLP-1R, GIPR, GLP-2R and GCGR were screened using a newly developed membrane-based split-ubiquitin yeast two-hybrid (MYTH) system to reveal 181 novel candidate protein interactors associated with signal transduction, transport, metabolism and cell survival. Each candidate was validated using yeast two-hybrid prey retransformation tests and by co-purification to confirm coupling to each receptors. The present work is the first demonstration of a split-ubiquitin interaction screen using in situ membrane expressed GPCRs of the secretin-like B class.

The objective of this thesis was to relate and link the physiological responses of the cells to the electrical responses or output obtained from the TSM acoustic wave sensor. In particular, the device was applied to the study of immortalized murine hypothalamic neurons (mHypoE-38 and -46 cell models) under a variety of conditions and stimuli. Cellular studies which lead to the production of detectable neuronal responses include neuronal deposition, adhesion and proliferation, alteration in the extent of specific cell-surface interactions, actin filament and microtubule cytoskeletal disruptions, effects of cell depolarization, solution tonicity, inhibition of the Na+-K+ pump via ouabain, effects of neuronal synchronization and the effects ligand-receptor interaction (glucagon). In addition, the introduction of drugs, neurotrophic factors (forskolin and beterferon), toxicity agents (NaOH, EtOH) and TiO2 nanoparticles were similarly investigated. A preliminary study conducted with mouse embryonic stem cells showed that not all cell lines are suitable for investigation with the TSM sensor at the current stage of sensor development. It has been found that control studies conducted with water as the solvent and the bare sensor substrate is insufficient to model the behavior of the sensor in the absence of cells. When biological buffers are used in addition to protein coatings the sensor responses are altered in magnitude and direction. To analyze the full range of cellular changes observed on the TSM sensor, the full impedance spectrum is required. As such in this thesis, the series and parallel resonant frequencies, the motional resistance, the maximum phase of the impedance and the static capacitance (fs, fp, Rm, θmax and Co were used to characterize the cellular responses observed. In the presence of cells fs shifts are largely influenced by the damping of the TSM resonator. The formation of cell-surface interactions and hence the increase in coupling and acoustic energy dissipation can be modeled as an additional resistor in the BVD model. Further sensor and cellular changes can be obtained by negating the effects of damping from fs with the use of Rm and θmax.

G-protein coupled receptors (GPCRs) have been shown to act as part of GPCR associated protein complexes (GAPCs) which are required to appropriately transduce downstream signaling pathways leading to specific cellular actions. I hypothesize that there are distinct molecular effectors that couple to the glucagon superfamily of B-class GPCRs (glucagon, GLP-1, GLP-2, GIP receptors) to effect the myriad of reported actions in numerous target cells including regulation of insulin secretion, intestinal growth and appetite suppression. GLP-1R, GIPR, GLP-2R and GCGR were screened using a newly developed membrane-based split-ubiquitin yeast two-hybrid (MYTH) system to reveal 181 novel candidate protein interactors associated with signal transduction, transport, metabolism and cell survival. Each candidate was validated using yeast two-hybrid prey retransformation tests and by co-purification to confirm coupling to each receptors. The present work is the first demonstration of a split-ubiquitin interaction screen using in situ membrane expressed GPCRs of the secretin-like B class.

La glycogénose de type 1a (GSD1a) est une maladie métabolique rare liée à une absence d'activité glucose‐6 phosphatase (G6Pase). La G6Pase est une enzyme clé de la production endogène de glucose (PEG) catalysant l'hydrolyse du G6P en glucose avant sa libération dans le sang. Cette fonction est restreinte au foie, aux reins et à l'intestin. La GSD1a est caractérisée par des hypoglycémies chroniques, une hépatomégalie associée à une stéatose hépatique et une néphromégalie. A plus longterme, la plupart des patients développent des adénomes. Un modèle murin de GSD 1a existe mais les souris ne survivent pas après le sevrage. Nous avons donc généré un modèle original de GSD1a, en invalidant le gène de la sous‐unité catalytique de la G6Pase spécifiquement dans le foie, grâce à une stratégie CRE‐LOX inductible (souris L‐G6pc‐/‐). Dans ce travail, nous avons montré que les souris L‐G6pc‐/‐ sont viables et reproduisent parfaitement la pathologie hépatique de la GSD1a, y compris le développement d'adénomes hépatiques après 9 mois d'invalidation. La viabilité des souris nous a permis de débuter des traitements par thérapie génique ciblant le foie à l'aide de vecteurs lentiviraux et AAV. La survie de ces souris, qui ne peuvent pas produire du glucose par le foie, repose la question du rôle relatif de la production hépatique de glucose dans la régulation de la glycémie Nous avons montré que les souris L‐G6pc‐/‐ sont capables de réguler leur glycémie, même au cours d'un jeûne prolongé. Ce maintien de l'homéostasie glucidique est due à une induction rapide de la néoglucogenèse rénale et intestinale, principalement par un mécanisme dépendant du glucagon

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Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography: 196-214.