1. The Effect of Creatine Monohydrate on Insulin Receptor Auto-Phosphorylation in H4IIE Rat Hepatoma Cells By Joe Gillen York College of Pennsylvania, Department of Biological Sciences Results Creatine Alone Creatine Pretreat + Insulin Control Ladder 12 hr 18 hr 24 hr 12 hr 18 hr 24 hr Insulin 210 125 IR 81 Figure 1: Effect of Creatine on Insulin Receptor Phosphorylation. Cells were serum starved then treated with 100 μM creatine alone 12-24 hr or in combination with 50 nM insulin 5 min. Isolated using a 7.5% SDS-PAGE gel and detected using PY-99 Mouse Anti-phosphotyrosine and Goat Anti-Mouse Horse Radish Peroxidase Conjugate with 4CN-OPTI. Creatine Alone Creatine Pretreat + Insulin Control 6 hr 21 hr 24 hr 6 hr 21 hr 24 hr Insulin Ladder 210 125 IR 81 Figure 2: Effect of Creatine on Insulin Receptor Phosphorylation. Cells were serum starved then treated with 100 μM creatine alone 6-24 hr or in combination with 50 nM insulin 5 min. Isolated using a 7.5% SDS-PAGE gel and detected using PY-99 Mouse Anti-phosphotyrosine and Goat Anti-Mouse Horse Radish Peroxidase Conjugate with 4CN-OPTI. Creatine Alone: - 6 hr treatment caused the same response as Insulin alone - 24 hr treatment caused a similar response as Insulin alone in one experiment - 12, 18, 21 hr treatments caused no response Creatine followed by Insulin: - 6 – 21 hr treatments caused a slight decrease in the Insulin response - 24 hr treatments blocked the insulin response in both experiments Literature Cited "Diabetes Mellitus." Encyclopædia Britannica. 2003. Encyclopædia Britannica Online. 28 Jul, 2003.http://www.search.eb.com/eb/article?eu=1569 Butler, A.E., Janson, J., et. al. “β-cell deficit and increased β-cell apotosis in humans with type 2 diabetes.” Diabetes 52 (2003): 102-110. Davis, C.M., Royer, A.C., and J.B. Vincent. “Synthetic multinuclear chromium assembly activates insulin receptor kinase activity: functional model for low- molecular-weight chromium-binding substance.” Inorganic Chemistry 36 (1997): 5316-5321. Earnest, C.P., Almada, A.L., and T.L. Mitchell. “High-Performance Capillary electrophoresis-pure creatine monohydrate reduces blood lipids in men and women.” Clinical Science 91 (1996): 113-118. Eijnde, B. O., Urso, B., Richter, E.A., Greenhaff, P.L. and P. Hespel. “Effect of oral creatine supplements on human muscle GLUT4 protein content after immobilization.” Diabetes 50 (2001): 18-23. Lee, Jongsoon and Paul F. Pilch. “The Insulin Receptor: Structure, Function, and Signaling.” American Physiological Society 94 (1994): C319-C334. Trischitta, V., Brunetti, A., et. al. “Defects in insulin-receptor internalization and processing in monocytes of obese subjects and obese NIDDM patients.” Diabetes 38 (1989): 1579-1585. Introduction The hormone insulin and its receptor regulate the transportation of carbohydrates into the cell. The body’s muscles are the main consumers of energy and made of individual cells, which metabolize carbohydrates to produce energy, are the main target of insulin in the body. When insulin comes in contact with the cell’s membrane it binds to its receptor causing the autophosphorylation of the tyrosines residues. This begins a cascade of reactions, which results in the translocation of GLUT4 to the cell membrane and the uptake of glucose from the blood surrounding the cells and lowers the glucose concentration in the circulating blood (Lee and Pilch 1994). Type II diabetes is characterized by high levels of blood glucose and an inability of the body’s own insulin to regulate its levels by absorbing glucose from the serum. The high concentration of glucose in the serum can lead to blindness and circulatory problems as glucose begins to harden in the blood (Britannica 2004). To treat this, doctors prescribe insulin regiments where insulin is injected into the body. However this insulin therapy eventually becomes ineffective and progressively larger doses of insulin must be used to regulate the growing glucose concentration. Eventually a limit is reached where to body no longer recognizes any insulin and glucose levels begin to rise unchecked. Chromium supplements have been shown to cause an increase in the activation of the insulin receptor in membrane fragments. However by adding monoclonal antibodies that block the insulin-binding site or removing insulin, the effect of chromium was inhibitited (Davis and Vincent 1997). Creatine supplements have also been shown to increase the activation of GLUT4 and raise the muscle cell bound glycogen in healthy individuals given as an oral supplement (Eijnde et al. 2001). However the mechanism for this decline in serum glucose concentration and rise in muscle bound glycogen is unknown. While the mechanism by which creatine functions is unknown, it appears to increase the activity of the insulin pathway because when creatine is added to a system insulin dependency and circulating blood glucose decrease. The goal of this study was to determine the effect of creatine on insulin receptor phosphorylation and insulin gene expression. Our study showed that after prolong treatment with creatine of 24 hours; the combination of creatine followed by insulin cause the down-regulation of the insulin receptor in Hepatoma cells. Methods Discussion: Insulin is secreted by the pancreas and used to regulate the concentration of glucose in the blood. Insulin activates the insulin receptor causing a signal cascade, which results in the absorption of glucose into the cells where it can be metabolized to produce adenine triphosphate (ATP) or glycogen can be produced. Having phosphates attach to tyrosines located on the internal side of the receptor activates the insulin receptor through autophosphorylation (Lee and Pilch 1994). In type II diabetes, blood glucose levels rise as food is metabolized however the body low sensitivity to insulin to cause the regulation of these levels. To treat this doctors prescribe insulin therapy if diet and exercise cannot control the rising levels of glucose. However over time insulin injections lose their effectiveness, and eventually the rise of glucose in the blood becomes uncontrollable (Butler et. al. 2003). This build up of glucose can lead to circulatory problems and blindness. Creatine monohydrate supplements showed a trend to reduce blood glucose level in healthy men and women (Earnest, Almada, and Mitchel 1996). Addition of creatine supplements to human subjects during a period of rehabilitation from an immobilized leg showed increased amounts of muscle bound glycogen and GLUT4 activity (Eijnde et al. 2001). Creatine showed an ability to down regulate the phosphoylation of tyrosine residues at the same molecular weight as the insulin receptor is found, and in the same pattern as only seen in the control cells given insulin, in the samples tested when the cells were treated for less than 24 hours. This pattern can lead us to believe that the band of phosphotyrosine in question was the autophosphorylated insulin receptor. This activation also decreased slightly as the exposure time to creatine increased. At 24 hours exposure the insulin receptor activation by insulin was blocked in both tests. Conversely at 6 hour exposure to creatine alone insulin receptor was activated. The same occurred once with 24 hour exposure to creatine alone however when that test was repeated there was no band so the effect is not reliable at best. How though can creatine cause the insulin pathway to down-regulate and eventually total shut down the receptor? When exposed to insulin for long periods of time, the insulin receptor eventually can become overactive. To limit the activity of the insulin receptor, the receptor will internalize, or change conformation so that the receptor portion of the molecule is inside the membrane. Once inside the membrane, the receptor cannot be activated by insulin (Trischitta et. al 1989). The down regulation of the insulin pathway in liver cells causes an increase in gluconeogenisis, or an increase in the amount of glucose made by the liver. This would slightly increase the amount of glucose in the blood. Since creatine is found naturally in the muscle cells, and since stressed cells have been known to lyse or have their membranes begin to leak slightly, the release of creatine into the blood could act as a cellular marker for the cells to signal that the muscle cells need glucose. To overcome the stressor muscle cells could be stimulated by the presence of creatine to begin drawing glucose from the blood in high concentrations, which would explain the significant increase in muscle bound glycogen as seen by Eijnde (2001). The increased production of glucose by the liver cells would be reasonable since the need for glucose would be very high. This study showed a relationship between creatine and insulin receptor phosphorylation, which should be further researched. 100 μM Creatine Treatment (0-24 hr) 50 nM Insulin (5 min) Cell Lysis: RIPA Buffer with Protease and Phosphotase Inhibitors BCA Assay Chemical Treatments Total Protein Quantification 7.5% SDS-PAGE Gel 50 µg Protein 170 V for 50 min Membrane Transfer: Nitrocellulose Membrane Towbin’s Buffer 10%MeOH 350 mA for 1 hr 1 st Antibody: PY99 Mouse Anti- phosphotyrosine Overnight at 4°C 2 nd Antibody: Goat Anti- Mouse – HRP Conjugate 45 min at Room Temp Horse Radish Peroxidase Assay (4CN-OPTI) 1 hr, Rocking at RT Western BlotDetection