1 The effect of thylakoids on body weight and appetite regulation Charlotte Erlanson- Albertsson and Per- Åke Albertsson The strong increase in the prevalence of overweight and obesity in recent years is one of the greatest threats to the health of the developed world (Fig. 1). Along with obesity there is an increased morbidity and mortality, the diseases being atherosclerosis, diabetes (Fig. 2), certain cancers, arthritis and Alzheimers disease. Global obesity Fig. 1. Obesity in the world. The highest frequency is in USA and in the Middle East, but is coming in Africa, South America, and China.!"#$%&'$'(%)*+*,$ Global diabetes Fig. 2. Frequency of diabetes in the world by 2025 and the correlation between obesity and diabetes. The highest frequency of diabetes is estimated to occur in Middle East and South America. Obesity has a strong impact on the development of diabetes, particularly in women. The cause of obesity is an imbalance between energy intake and energy expenditure. The energy intake is considered to be the most important factor in this balance. In parallel with the rapid rise in obesity there has been an increased consumption of calories, in USA by 530 kcal per day since 1970 and in Sweden by 287 kcal since 1980. Even a modest weight loss can reduce the various consequences of obesity. At present time gastrointestinal surgery offers the main solution to overweight and obesity. Since these operations may induce intestinal problems it is important to find other non- surgical solutions to the question of obesity. The positive results with gastrointestinal surgery have been explained in part by the change in the release of gut hormones and their influence on appetite regulation.
2 Appetite control is hence crucial. Appetite regulation occurs through an interplay between gut hormones and the brain. Ghrelin is an important hunger signal released from the stomach, triggering eating and also the formation of fat in adipose tissue. Cholecystokinin (CCK) and Glucagon- like peptide 1 (GLP- 1) are two important satiety hormones, known to promote satiety in humans. As a result there has been an increased interest to use these hormones as anti- obesity drugs. Such gut hormone administration has been demonstrated to be an effective anti- obesity therapy. The difficulties lie in the way of administration, since these hormones are easily destroyed and cannot be given orally. Our discovery concerns the use of thylakoids to release endogenous satiety hormones, such as CCK and GLP- 1. Thylakoids thus release anti- obesity hormones from the body. Thylakoids could be added to food and consumed in small amounts. During normal appetite control gut satiety hormones are released and act centrally to stop eating (Fig. 3).!,((-&./&/+(0+1&-.2#/%#-&.)& %3#&.)%#/$)#&!""#$%#&'()%*(+& Fig. 3. Normal appetite regulation. Satiety hormones are released from the gut and acting in the brain to stop eating and give satisfaction.! 45%&3(*6()#/&7*#& *#+#7/#-&%(&2.8#&/7$#%1& 97$#%1& Gut hormones The problem today is that food is too easily digested and absorbed from the intestine, hence failing to give enough of satiety hormones. There is also too little food to be digested in the colon, due to lack of fibres and pigments. A good bacterial flora in the large intestine is needed for optimal appetite control and health. A third problem is the blood glucose changes with modern food, which trigger appetite and hunger (Fig. 4). Dysregulation of appetite!!"#$%&'(""#')*'+""'%,-)#./'#)0$*+$#',&#',1*"%1$#''! 2"3'.$4$.*'"('05+'*,6$+/'7"%8"&$*'! 2"3'.$4$.*'"('0""#'1,9+$%),')&'+7$'9"."&'! :%,8,69'1.""#'*50,%'97,&0$*' Fig. 4. Dysregulation of appetite occurs today with the consumption of modern food. The food is too rapidly digested and absorbed. An empty stomach is hungry. The whole intestine needs to work for production of satiety signals. Also the large intestine needs to have food for the production of the health promoting bacteria. We are not made for dramatic blood glucose changes as occurs with modern food.
3 Thylakoids retard food digestion. The start of thylakoids for use as anti- obesity therapy was based on the finding that thylakoids retard fat digestion. The actual idea that came up was to find something natural that contained galactolipids known to retard fat digestion. Thylakoids contain galactolipids. And indeed we found that thylakoids retarded fat digestion (Fig. 5). Fat digestion occurs normally by the activity of two proteins, pancreatic lipase and colipase. When thylakoids are mixed together with liquid fat they bind to the surface of the lipid droplets thereby hindering the pancreatic lipase/colipase complex to interact with the lipids. In addition thylakoids bind to lipase/colipase. However since the thylakoids are themselves composed of proteins and lipids they will be broken down in the gastrointestinal tract. The net result is that fat digestion is just retarded. Eventually the fat is taken up by the intestine without any fatty stools. It is of interest that our project started with the idea to find something natural that contained galactolipids known to retard fat digestion. However, it turned out that it was mainly the proteins of the thylakoids and not the galactolipids, which were responsible for the retardation of fat digestion by specific interactions with lipase/colipase and the fat droplets. Thylakoids retard fat digestion 100 Activity Lipase/colioase (%) 90 80 70 60 50 40 30 20 10 0 0 0,5 1 1,5 Thylakoids Control Colipase Lipase Lipase Lipas Colipase Thylakoids Amount thylakoid membranes (mg chlorophyll) Triacylglycerol Triacylglycerol Fig. 5. The addition of thylakoids retards fat digestion. The explanation is the binding of thylakoids to the oil droplet and the binding of thylakoids to pancreatic lipase and colipase. What are thylakoids? Thylakoids are membranes that are responsible for the light reaction of photosynthesis in green plant cells (Fig. 6). They are situated in certain organelles called chloroplasts that lie close to the cell wall in the border of the cell.
4 Green plant cell Thylakoids in chloroplast Thylakoids!"#$%$&#'()(*+,)"*)"-#'.$,/(*!0##*+'##* Nucleus Fig. 6. Green plant cell with chloroplasts (left) and an enlarged chloroplast (right) with thylakoids (right). The thylakoids are membranes which form stacked structures. They contain several protein, lipids, antioxidants and pigments. There are two large protein/pigment complexes, named Photo system I and II (PS I and PS II), which bind all the chlorophyll of the thylakoids (Fig. 7). These two photo systems absorb sun light and convert light energy into chemical energy used for synthesis of glucose and starch in the chloroplast and other components of the plant cell. In addition, PS II splits water into air oxygen and protons. Since sun energy and oxygen are is potentially harmful to the plants there are also various antioxidants and vitamins to protect the plant cell.!"#$%$&"'##( Fig 7. Thylakoids and their role in photosynthesis. Left: The thylakoid membrane with its main components working in the light reaction in photosynthesis: PSII takes up light from sun and splitts water into oxygen and protons. Plastoquinone (PQ), cytochrome b 6f and plastocyanin transport electrons from PSII to PSI. ATPsynthase uses the transport of protons to produce ATP, released on the stroma side of the thylakoid membrane. Right: Molecular structure of one light harvesting complex (PSII) showing the spiral protein chains together with the bound pigments chlorophyll and carotenoids.
5 Thylakoids are prepared by mincing green leaves into small pieces followed by filtration to remove large particles and cell walls. The filtrate, which contains the thylakoids are then precipitated with citric acid and collected by centrifugation. The precipitate is washed with water and then freeze- dried to a powder. Out of 100 g spinach around 5 g powder is achieved (Fig. 8). The composition of 100 g thylakoids is described in Table I. Fig. 8. Preparation of thylakoids from spinach gives a green powder. Out of 100 g spinach 5 g of thylakoids is obtained. This is the daily dose that has in human studies been shown to reduce body weight and suppress hunger. Table I Composition of 100 g thylakoid powder Substance Quantity Substance Quantity Substance Quantity Protein 23,5 g Vitamin A 21 ug Betakaroten 4760 ug Fat 11,9 g Folic acid 166 ug Lutein 27,9 mg Carbohydrate 41,7 g Vitamin K 1313 ug Zeaxantin 730 ug Chlorophyll 3000 mg Satiety promoting effect of thylakoids in human was demonstrated first using thylakoids in a pesto sandwich, eaten together with basil, walnuts, tomatoes and coffee as a breakfast. The study was a cross- over study with healthy normal weight subjects, where the subjects served as control and tried various doses of thylakoids. The satiety hormone CCK was significantly raised during six hours after start of the meal compared to a control meal, which raised the satiety hormone CCK for only two hours (Fig. 9).
6 Serum CCK (pmol/l) 2,5 2,0 1,5 1,0 Control Thylakoids 0,5 0,0-50 0 50 100 150 200 250 300 350 400 Time (min) Fig. 9. Thylakoids in a pesto on a bread served for breakfast (left) caused the release of the satiety hormone CCK in healthy humans in a significant way compared to control (right). Cholecystokinin is a satiety hormone that slows down movement of the intestine, ensuring everything to be absorbed by the intestine. It also acts centrally in the brain to release serotonin and dopamine from nucleus accumbens to promote a reward, stability and satisfaction with the amount of food eaten (Fig. 10). There is a lower uptake of fatty acids into the blood, suggesting that fatty acids may be oxidised already in the intestine in the presence of thylakoids.! Slow digestion of fat! Release CCK!"#$%&'()*+,%-./#+ 0.1%2)+ Fig. 10. Thylakoids slow down fat digestion, and raise the satiety hormone cholecystokinin (CCK). This hormone acts both in the intestine to slow down intestinal motility and in the brain to promote satisfaction by release of serotonin and dopamine in the brain. CCK
7 Table II Qualitative description of thylakoids Substance Quality Substance Quantity Protein 100 different Provitamin Betakaroten Fat Galactolipids (80%) Phospholipids (10%) Sulpholipids (10%) Vitamins Vitamin A Vitamin E Vitamin K1 Folic acid Carbohydrate Cellulose Antioxidants Lutein Violaxanthin Zeaxanthin Pigments Chlorophyll a and b Plastoquinon The suppression of hunger by thylakoids has been demonstrated after a single meal. This meal could contain either a high amount of fat or a high amount of carbohydrate. This means basically that thylakoids suppress appetite irrespective of the composition of the meal. The hunger suppression is most intense between 4 and 6 hours after the start of meal (Fig. 11). The hunger hormone ghrelin is suppressed by thylakoids more than in a control group. VAS Hunger (mm) 60 40 20 Control Thylakoid 0 0 30 60 90 120 150 180 210 240 270 Time (min) * Fig. 11. Suppression of hunger and thoughts of food with thylakoids compared to control. At time point zero 5 g of thylakoids was eaten with a high carbohydrate breakfast containing bread, butter, cheese, juice, yoghurt, musli and marmalade. The thylakoids were mixed with the marmalade. Hunger was scored during four hours. Control subjects received marmalade only. The effect of thylakoids to suppress hunger started after 2 hours and became significant after four hours. The suppression of wish for sweet by thylakoids was demonstrated in overweight women in single meal experiments. A high- carbohydrate breakfast was given with and without thylakoids (5 g) in a blueberry shot, where after the wish for sweet was estimated. After 180 minutes the wish for sweet items started and was high at time point 360 min, when lunch was served. Time point 180 min corresponds to the time when blood glucose is below fasting value and this is the cause for craving of sweet. With thylakoids there was a strong suppression of wish for sweet during the six hours studied. How could the thylakoids stop wish for sweet? Wish for sweet is a sign of energy deficiency. The cells in the brain lack glucose. The stop for such sweet craving means
8 that the cells have glucose and do not crave for it. This is achieved through the hormones that trigger the use of glucose as energy substrate. One such important hormone is glucagon- like peptide 1. This is released from the gut and has both intestinal and central effects. In the intestine it slows intestinal motility and in the brain it gives a satiety and satisfaction, especially related to sweet. Glucagon- like peptide releases insulin, which in turn stimulate the use of glucose as energy substrate. Glukagon- like peptide stops craving for sweet. Thylakoids release gut hormones that stop the craving for sweet. There are probably several explanations for the suppression of craving for sweet by thylakoids. One explanation is the release of glucagon- like peptide, which is significantly raised by thylakoids compared to control (Fig. 12). Another explanation may be the suppression of ghrelin, which is significantly suppressed by thylakoids compared to control. A reduced craving for sweet is very important to achieve appetite control.!"#$%&'()'*)$+,")*'+)-.""/)!! Thylakoids reduce urge for sweet! Gut homone GLP-1 is released 0')$+,")*'+) -.""/) Fig. 12. Thylakoids reduce the urge for sweet. This occurs through the release of glucagon- like peptide from the gut, which stops the urge for sweet taste. GLP-1 Thylakoids are prebiotic. The intestinal microflora is considered important for health. Some bacteria are health- promoting, preventing various disease conditions such as obesity and diabetes. One healthy strain of bacteria is Lactobacillus Reuteri. This has been found to be overrepresented in lean subjects compared to obese subjects, suggesting them to promote thinness. We have found that thylakoids when given to rat for fourteen days in the diet change their gut microflora, with an increase of Lactobacillus Reuteri in the distal intestine (Fig. 13). These observations suggest that thylakoids are prebiotic, which means that they change the bacterial flora in a health promoting way.!"#$%&'()#*#(+)&,)+-./01&%23) Lactobacillus Reuteri Fig. 13. Thylakoids promote the colonization of healthy bacteria in the intestine, like the Lactobacillus Reuteri. They are identified in the distal intestine, ileum, but not in the colon, in rat after 14 days of feeding with thylakoids.
9 Thylakoids promote body weight loss and reduce blood lipids after 8 weeks of daily treatment. Studies in overweight subjects have demonstrated that thylakoids when given daily in a 5 g dose prior to breakfast reduce body weight, blood lipids, blood cholesterol, fat mass and leptin, and change the micro flora. During these studies meals were restricted to three times daily to optimize the effect of thylakoids, since they produce a long- lasting satiety (Fig. 14). Leptin in blood was reduced as was total fat, truncal fat and blood lipids compared to control. Leptin is secreted from fat mass. A reduction of leptin levels in the blood suggests that fat mass has been lost. A loss of total fat and truncal fat was measured through impedance measurements. The reduction of blood lipids suggest that fat is being used as energy substrate. Thylakoids: loss of fat mass Leptin Blood lipids Control Thylakoids Total fat Truncal fat!"#$#%& Fig. 14. Loss of fat mass with thylakoids during 8 weeks in overweight women compared to control. All subjects were instructed to eat three times daily and exercise 30 min daily. Thylakoids in total 5 g were consumed as a blue berry shot. The control group consumed a blueberry shot every day without thylakoids. Summary Ten main effects have been demonstrated for thylakoids Promotion of satiety Dampening of hunger Reduction of body weight Reduction of fat mass Reduction of truncal fat mass Reduction of blood lipids Reduction of serum cholesterol Stabilization of blood glucose Decreased urge for sweet Pre- biotic effect on intestinal bacterial flora References: Thylakoids; studies in human: 1. Köhnke R, Lindbo A, Larsson T, Lindqvist A, Rayner M, Emek SC, Albertsson PA, Rehfeld JF, Landin- Olsson M, Erlanson- Albertsson C. Thylakoids promote release of the satiety hormone cholecystokinin while reducing insulin in healthy humans. Scand J Gastroenterol. 2009; 44(6):712-9. 2. Stenblom E- L, Montelius C, Östbring K, Håkansson M, Nilsson S, Rehfeld JF and Erlanson- Albertsson C. Supplementation by thylakoids to a high carbohydrate meal
10 decreases feelings of hunger, elevates CCK levels and prevents postprandial hypoglycaemia in overweight women. Appetite 2013; Sep 68: 118-23. 3. Montelius C, Stenblom E- L, Skarping L, Fransson M, Erlandsson D, Podgorski K and Erlanson- Albertsson C. Loss of body weight and fat mass in overweight women coupled to suppressed hunger after two months diet intervention with thylakoids from spinach. Submitted for publication (2014). 4. Montelius C, Erlandsson D, Vitija E, Podgorski K and Erlanson- Albertsson C. Decreased urge for sweet coupled to increased GLP- 1 levels in overweight women after three months diet intervention with thylakoids from spinach. Manuscript in preparation (2014). Thylakoids; animal studies, biochemistry: 5. Albertsson PA, Köhnke R, Emek SC, Mei J, Rehfeld JF, Akerlund HE, Erlanson- Albertsson C. Chloroplast membranes retard fat digestion and induce satiety: effect of biological membranes on pancreatic lipase/co- lipase. Biochem J. 2007; Feb 1;401(3):727-33. 6. Köhnke R, Lindqvist A, Göransson N, Emek SC, Albertsson PA, Rehfeld JF, Hultgårdh- Nilsson A, Erlanson- Albertsson C. Thylakoids suppress appetite by increasing cholecystokinin resulting in lower food intake and body weight in high- fat fed mice. Phytother Res. 2009; Dec;23(12):1778-83. 7. Montelius C, Osman N, Weström B, Ahrné S, Molin G, Albertsson P- Å and Erlanson- Albertsson C. Feeding spinach thylakoids to rats modulates the gut microbiota, decreases food intake and affects the insulin response. Journal of Nutritional Science 2013; vol. 2, e20 p1-9. 8. Emek SC, Szilagyi A, Akerlund HE, Albertsson PA, Köhnke R, Holm A, Erlanson- Albertsson C. A large scale method for preparation of plant thylakoids for use in body weight regulation. Prep Biochem Biotechnol. 2010;40(1):13-27. 9. Rayner M, Ljusberg H, Emek SC, Sellman E, Erlanson- Albertsson C, Albertsson PA. Chloroplast thylakoid membrane- stabilised emulsions. J Sci Food Agric. 2011 Jan 30;91(2):315-21. 10. Montelius C, Gustafsson K, Weström B, Albertsson PÅ, Emek SC, Rayner M, Erlanson- Albertsson C. Chloroplast thylakoids reduce glucose uptake and decrease intestinal macromolecular permeability. Br J Nutr. 2011 Sep;106(6):836-44. 11. Emek SC, Akerlund HE, Clausen M, Ohlsson L, Westrom B, Erlanson- Albertsson C, Albertsson PA. Pigments Protect the Light Harvesting Proteins of Chloroplast Thylakoid Membranes Against Digestion by Gastrointestinal Proteases. Food Hydrocolloids 2011;25(6): 1618-1626. 12. Montelius C, Szwiek K, Kardas M, Lozinska L, Erlanson- Albertsson C, Piwezynowski S, Rehfeld JF and Weström B. Dietary thylakoids suppress blood glucose and modulate appetite- regulating hormones in pigs exposed to oral glucose tolerance test. Clinical Nutrition. 2014, doi:10.1016/j.clnu.2013.12.009.