Department of Anatomy and Neurobiology University of Maryland School of Medicine 20 Penn St. HSFII / Rm S251 Baltimore, MD 21201
M.Geraedts@umaryland.edu
Maartje Geraedts was born on March 4th 1982 in Asselt, The Netherlands. She completed secondary school at the “ Bischoppelijk College Broekhin” in 1998. In the same year, she started her study medical microbiology at the Gilde Opleidingen (Blerick, The Netherlands), where she graduated in 2001. She started her study Biochemistry at the Hogeschool Arnhem en Nijmegen (Nijmegen, The Netherlands) in 2001, followed by the study Master of Molecular Life Science in 2004 at the same location, where she graduated in 2005. She did her master thesis entitled ‘Glycosaminoglycans in the diaphragm of COPD patients’ at the department of Pulmonary Diseases, Radboud Medical Center Nijmegen. In February 2006, Maartje started her PhD at the University of Maastricht, at the department of Human Biology. She studied the effects of dietary proteins on release of satiety hormones from gastrointestinal cells, under supervision of Prof. Wim H.M. Saris and Dr. Freddy J. Troost. The research performed in this period is described in this thesis. In 2009, she was awarded a 3th place ‘Foppe en Hoor’ young investigator award by the Netherlands Organisation of Scientific Research (NWO) for her work on the effects of intraduodenally administered pea protein on food intake in lean and obese subjects. Currently, Maartje is working as a post-doc at the department of Anatomy and Neurobiology, University of Maryland School of Medicine, USA.
Abstract: Scope: Consumption of high-protein diets cause elevated levels of CCK and GLP-1. Although unknown, this might be due to protein breakdown by various proteases that originate from the gastrointestinal tract. This study investigated which dietary proteins, hydrolysates, or synthetic-peptides are most potent to affect secretion of CCK and GLP-1 in STC-1 cells known for satiety hormone release. Methods and results: Addition of intact proteins to STC-1 cells exerted strong effects on secretion of satiety hormones. Casein, whey, and pea showed strongest effects on CCK release, whereas casein, codfish, egg, and wheat showed most pronounced effects on GLP-1 release. Egg-hydrolysate stimulated release of CCK and GLP-1, whereas all other tested hydrolysates and synthetic-peptides showed no significant effects on hormone release. Addition of a combination of trypsin and casein-hydrolysate, codfish, egg, egg-hydrolysate, sodium-casein, wheat-hydrolysate, or wheat resulted in additional stimulation of CCK release, compared to only the protein. Addition of a combination of DPP-IV and egg-hydrolysate, ovomucoid, or sodium-casein decreased GLP-1 levels. Conclusion: This study showed that specific intact, or partially digested proteins, in contrast to protein-hydrolysates and synthetic-peptides, stimulated hormone release. We conclude that intact proteins exert strong effects on satiety hormone release, and may therefore provide potent dietary supplements for prevention or treatment of obesity.
Abstract: High protein diets are the most effective to stimulate cholecystokinin (CCK) and glucagon-like peptide 1 (GLP-1) release; however, which proteins are the most potent is not known. Here, the effects of specific dietary proteins on intestinal CCK and GLP-1 release were examined.
Abstract: This review discusses the role of enteroendocrine cells in the gastrointestinal tract as chemoreceptors that sense intraluminal contents and induce changes in food intake through the release of signalling substances, such as satiety hormones. Recent evidence supports the concept that chemosensing in the gut involves G protein-coupled receptors (GPCRs) that are known to mediate gustatory signals in the oral cavity. GPCRs can be grouped into several families, depending on the stimuli to which they respond, e.g. proteins, amino acids, carbohydrates, fatty acids, or tastants. Sensing of these stimuli by GPCRs results in hormone secretions of enteroendocrine cells, which participate in the control of food intake. A better understanding of the stimuli that induce the strongest binding with these receptors, and thus induce a strong release of hormones, can be a very useful strategy for the development of novel foods in the treatment of obesity.
Abstract: Dietary modulation of the response of gut satiety hormones, which partly regulate food intake, provides a promising treatment for overweight and obesity. Gut-derived cell lines such as STC-1 are widely used to investigate these hormonal responses to nutrients. To date, no peptide-YY (PYY) secreting cell line has been identified. The aim of this study was to investigate whether STC-1 cells are able to secrete PYY and if so, whether dietary compounds can modulate PYY secretion. The effects of fatty acid types C4:0, C12:0, C14:0, C16:0, and C18:0 on PYY release were investigated by measuring PYY in the supernatant after 30, 60, 90, and 120 min of incubation, respectively, using RIA assays. The STC-1 cells were able to secrete PYY in a time-dependent manner. It was shown that after 30 min, C4:0, C12:0, C16:0, and C18:0 caused increased PYY levels compared to the control. At time points 60 and 90 min, C4:0 and C18:0 induced elevated PYY levels compared to the control. After 120 min, C4:0, C14:0, and C18:0 caused elevated levels compared to the control. We are the first to show that the STC-1 cells are also able to secrete PYY next to cholecystokinin (CCK) and glucagon-like peptide 1 (GLP-1). Addition of fatty acids resulted in increased levels of PYY, which is consistent with the literature describing human studies. We conclude that the STC-1 cell line provides an appropriate cell line for screening the effects of ingredients on the release of the satiety-related gut hormones CCK, GLP-1, and PYY.
Abstract: In the present study, phage display-derived antibodies were used to investigate the topology of glycosaminoglycan epitopes in the diaphragm of chronic obstructive pulmonary disease (COPD) and non-COPD patients. Furthermore, the potential physiological significance of changes in the occurrence of glycosaminoglycan epitopes in the diaphragm of COPD patients was studied by determining the overlap in epitope recognition of glycosaminoglycan antibodies and growth factors. Diaphragm cryosections from non-COPD (n = 5) and COPD patients (Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage I/II; n = 9) were incubated with antibodies directed against heparan sulphate, chondroitin sulphate and dermatan sulphate epitopes. Antibodies were visualised immunofluorescently. In addition, interference of antibody and growth factor binding with heparan sulphate epitopes was tested. Specific glycosaminoglycan epitopes showed increased expression in the diaphragm of COPD patients, whereas other epitopes were decreased or unaffected. Interestingly, the anti-heparan sulphate antibody HS4C3, which is directed against a downregulated epitope, interfered with the binding of hepatocyte growth factor. Three patients with the most severe airway obstruction also demonstrated interference of heparan sulphate antibody A04B08 with hepatocyte growth factor binding. Results indicate changes in glycosaminoglycan composition in the diaphragm of patients with chronic obstructive pulmonary disease. This may affect cellular physiology via alterations in growth factor handling and might be related to reduced levels of contractile protein in the diaphragm of these patients.
Abstract: Hypoxia impairs skeletal muscle function, but the precise mechanisms are incompletely understood. In hypoxic rat diaphragm muscle, generation of peroxynitrite is elevated. Peroxynitrite and other reactive nitrogen species have been shown to impair contractility of skinned muscle fibers, reflecting contractile protein dysfunction. We hypothesized that hypoxia induces contractile protein dysfunction and that reactive nitrogen species are involved. In addition, we hypothesized that muscle reoxygenation reverses contractile protein dysfunction. In vitro contractility of rat soleus muscle bundles was studied after 30 min of hyperoxia (Po2 approximately 90 kPa), hypoxia (Po2 approximately 5 kPa), hypoxia + 30 microM N(G)-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor), hyperoxia + 30 microM L-NMMA, and hypoxia (30 min) + reoxygenation (15 min). One part of the muscle bundle was used for single fiber contractile measurements and the other part for nitrotyrosine detection. In skinned single fibers, maximal Ca2+-activated specific force (Fmax), fraction of strongly attached cross bridges (alphafs), rate constant of force redevelopment (ktr), and myofibrillar Ca2+ sensitivity were determined. Thirty minutes of hypoxia reduced muscle bundle contractility. In the hypoxic group, single fiber Fmax, alphafs, and ktr were significantly reduced compared with hyperoxic, L-NMMA, and reoxygenation groups. Myofibrillar Ca2+ sensitivity was not different between groups. Nitrotyrosine levels were increased in hypoxia compared with all other groups. We concluded that acute hypoxia induces dysfunction of skinned muscle fibers, reflecting contractile protein dysfunction. In addition, our data indicate that reactive nitrogen species play a role in hypoxia-induced contractile protein dysfunction. Reoxygenation of the muscle bundle partially restores bundle contractility but completely reverses contractile protein dysfunction.
Abstract: COPD is associated with an increased load on the diaphragm. Since chronic muscle loading results in changes in antioxidant capacity and formation of reactive oxygen and reactive nitrogen species, we hypothesized that COPD has a similar effect on the diaphragm, which is related to the severity of COPD. Catalase activity was determined spectrophotometrically. Levels of 4-hydroxy-2-nonenal (HNE)-protein adducts and 3-nitrotyrosine (NT) formation were measured using western blotting. Levels of malondialdehyde (MDA) were assessed by high-performance liquid chromatography. We found that catalase activity was approximately 89% higher in the diaphragm of severe COPD patients (FEV1 37+/-5% predicted) compared with non-COPD patients. MDA levels, a marker for lipid peroxidation, were significantly lower in the diaphragm of COPD patients compared with non-COPD patients, whereas the level of HNE-protein adducts was equal in both groups. NT formation was not different between groups. However, increasing hyperinflation and NT formation were inversely correlated. These results indicate that in COPD the diaphragm adapts to a higher work load by increasing catalase activity, resulting in a reduction in oxidative damage to lipids and tyrosine nitration of proteins.
Abstract: Overweight and obesity have become a major health problem and strategies for prevention and treatment are needed. The most effective treatment is to decrease food intake, thereby modulating appetite sensations. The concept of appetite includes at least two separate components: satiation and satiety. Satiation refers to the processes that promote meal termination, thereby limiting meal size. Satiation results from signals that are produced in the gut in response to food-induced mechanical and chemical stimuli, such as the release of the satiety hormones cholecystokinin (CCK), glucagon-like peptide 1 (GLP-1), and peptide YY (PYY). These hormones are secreted upon food intake, since the gastrointestinal tract contains chemosensors that sense lumen contents and induce changes in gastrointestinal function and food intake though the release of these satiety hormones. Not only the macronutrient composition of the diet plays an important role in the release of these satiety hormones, but also the compounds, such as sweeteners, used to add flavour to the food affect hormone release. From the macronutrients, it seems that protein has the highest satiating effect when compared to other macronutrients in humans and animal studies. However, proteins differ in their amino acid composition and metabolism and thus may influence the satiating effects differently. There are also indications that tastants are able to affect hormone release from enteroendocrine cells, but whether this is true for all tastants, and whether there are differences between tastants is still unknown. In the present thesis several techniques are used to identify proteins and tastants that affected the gastrointestinal satiety hormone release the strongest.
The first technique that was used was the in vitro cell culture system. The STC-1 cell line, derived from an intestinal tumor arising in double transgenic mice, is known to secrete CCK and GLP-1 after exposure to nutritional compounds. STC-1 cells were incubated with several different proteins, hydrolysates, and synthetic peptides, and CCK and GLP-1 release was measured. We demonstrated that intact proteins such as casein, whey, and pea showed strongest effects on CCK release, whereas casein, codfish, egg, and wheat showed most pronounced effects on GLP-1 release. Egg-hydrolysate stimulated release of CCK and GLP-1, whereas all other tested hydrolysates and synthetic-peptides showed no significant effects on hormone release. As a second technique, the Ussing chambers were used. These chambers allow fresh human or animal tissue to stay alive for approximately 3-4 hours. The luminal side of both rat and human duodenal tissue was exposed to egg protein, codfish protein, pea protein, ovomucoid, or wheat protein for 2 hours. CCK and GLP-1 release was measured at the serosal side. Remarkably, no effects on CCK and GLP-1 release were found using rat duodenal tissue, whereas addition of pea and wheat protein did affect CCK and GLP-1 release from human duodenal tissue.
Overall, intact pea protein seemed to be the most potent protein to stimulate hormone release from duodenal tissue; however, this had to be confirmed in a human intervention study. The study described in the present thesis compared the effects of intact pea protein on satiety hormone levels and food intake after either oral ingestion or duodenal administration in both lean and obese subjects. We showed that food intake was decreased after intraduodenal protein administration in both lean and obese subjects, in contrast to food intake after oral ingestion of the protein. Although there were no differences in appetite ratings between both groups, we observed elevated levels of CCK in obese subjects, and GLP-1 and PYY were elevated in both groups. These data suggest that the degradation of intact pea protein in the stomach must be prevented in order to decrease food intake. Additionally, the results from this in vivo intervention study were compared to results obtained from Ussing chamber experiments, using the same volunteers. It was demonstrated that the trend in hormone release measured in the Ussing chambers was comparable with the trend that was observed in the intervention study, indicating that the Ussing chamber technology provides a valid screening tool to study the effects of nutritional compounds on hormone release.
Not only the effects of proteins, but also the effects of several tastants were tested on their effects on hormone release from STC-1 cells. All tastants increased CCK levels both dose- and time-dependently. GLP-1 release dose-dependently increased after addition of all tastants, with the exception of citric acid. GLP-1 was released in a time-dependent manner after addition of all tastants, but bitter tastants stimulated GLP-1 release only during the first 15 minutes of exposure. All commercial sweeteners elevated CCK and GLP-1 levels, with Tagatesse containing sucralose exerting the strongest effects.
Since sweeteners seemed to be the most potent tastants to stimulate hormone release, it might be possible that when combining sweeteners with intact pea protein, synergistic effects on hormone release may occur. Exposure to aspartame, sucralose, sucrose, pea, and pea with sucralose secreted elevated levels of CCK, whereas GLP-1 levels were increased after addition of all test-products. However, addition of sucrose and sucralose to human duodenal biopsies did not affect CCK and GLP-1 release; addition of pea did stimulate CCK and GLP-1 secretion. Combining pea protein with sucrose and sucralose induced even higher levels of CCK and GLP-1.
Overall, these studies show that infusing intact pea protein into the duodenum has the strongest effects on plasma hormone levels, and has the greatest effect on food intake. It has also been demonstrated that the combinations of dietary compounds may provide a novel dietary tool to adjust appetite and food intake. However, more studies will have to be performed to identify the lowest effective dose of the protein and sweetener, and whether (micro-) encapsulated proteins show the same effects on food intake. Also, long-term intervention studies will have to be performed to demonstrate the effects of intraduodenal pea protein administration on weight loss and weight maintenance.
Abstract: The invention is in the field of weight management, in particular in the field of weight management by influencing the mechanisms of body-weight regulation. Intact pea protein and intact wheat protein were found to be effective in reducing appetite or inducing or increasing satiety when brought into contact with their receptors in the duodenum. Since it is known that intact proteins hydrolyse in the gastrointestinal tract, intact pea protein and intact wheat protein will not exhibit their satiating effect when ingested in a conventional oral preparation. Therefore, special care should be taken to deliver the intact proteins to the duodenum in order for them to arrive there intact. One object of the invention may therefore be achieved by incorporating the intact protein in an enteric delivery vehicle.
