Background
Type II diabetes mellitus is a metabolic disorder found in adults that involves high blood sugar (hyperglycemia) due to insulin resistance as well as an insufficient amount of insulin. Type II diabetes differs from Type I diabetes, which is characterized by a complete lack of insulin. Symptoms of Type II diabetes include thirst, frequent urination and hunger. Long term complications include heart disease, strokes, vision problems and kidney failure. Today, there are close to 300 million people in the world who have been diagnosed with type II diabetes, an increase of 10 fold over the past 30 years. This increase in the incidence of diabetes is in parallel to the increase in the rate of obesity throughout the world, as well as an increase in the aging population. Type II diabetes is found in populations of the developed and developing world (ADA, 2014).
The vast majority of diabetes cases are Type II, or early onset diabetes. The development of Type II diabetes can be attributed in part to lifestyle as well as genetic factors. This disease can be managed by increasing exercise and making dietary changes in order to maintain a healthy weight. If these lifestyle changes cannot by themselves lower blood sugar levels, then medications may be required. The most frequently used medications are insulin or metformin. For those who take insulin, there is typically the requirement to routinely check blood sugar levels (WebMD, 2014).
The mechanism behind Type II diabetes is as follows: an insufficient amount of insulin is produced from beta cells (a subset of cells found in the pancreas that secrete the hormone insulin). This can lead to insulin resistance, meaning that cells are unable to respond to normal levels of insulin that are secreted from the beta cells. Insulin resistance can be the result of a reduced number of insulin-specific receptors on the surface of cells, leading to an inability to respond to insulin that is circulating in the bloodstream. Under normal circumstances, the liver suppresses insulin release, but for a Type II diabetes patient, the liver releases an improper level of glucose into the bloodstream. It is the combination of beta cell dysfunction and insulin resistance which results in the pathology of diabetes (Mayo Clinic, 2014).
If diet and lifestyle fail to prevent the onset of Type II diabetes, then externally applied insulin may be required. More than 40% of Type 2 diabetes patients use insulin to manage their diabetes. Human insulin is a protein of 58 amino acids and is well conserved among many living species. Recombinant insulin is administered subcutaneously via syringes or insulin pens. Metformin represents another popular medication that is prescribed for diabetes and is recommended by the American Diabetes Association. Unlike insulin, which becomes degraded in the GI tract if taken orally, metformin is administered in the form of a pill, and when taken as recommended, poses few significant risks for patients. Metformin is believed to act by suppressing glucose production in the liver, thus reducing hyperglycemia (high blood pressure). Metformin also increases insulin sensitivity and improves insulin binding to its receptor on the surface of cells (ADA, 2014).
Besides the use of medications, altering specific metabolic pathways can be a method to address diabetes as a disease. For example, the gluconeogenesis (meaning synthesis of new glucose) pathway results in producing glucose from a substrate that is a non-carbohydrate, such as pyruvate, lactate, glycerol, and glucogenic amino acids, as well as odd-chain and some even-chain fatty acids. Another method of generating glucose known as glycogenolysis, involves the degradation of glycogen and will not be discussed here.
Gluconeogenesis is a metabolic process that is found in many living species and can take place in the liver, the cortex of the kidneys and possibly in the intestine. In humans, gluconeogenesis takes place during fasting, on low-carbohydrate diets, or from intense physical activity. Gluconeogenesis is an alternative way for the body to generate glucose from noncarbohydrate sources such as amino and fatty acids. The liver uses primarily lactate, alanine and glycerol as substrates. The kidneys, on the other hand, use lactate, glycerol and glutamine as substrates. Lactate, for example, that is returned to the liver can be converted by lactate dehydrogenase into pyruvate, a substrate for the gluconeogenic pathway, which is used to generate glucose. Similarly, amino acids can undergo transanimation or deanimation to be converted into pyruvate or oxaloacetate, and thus become substrates for the gluconeogenesis pathway. The end result is the overproduction of glucose that can be characteristic of diabetes.
Gluconeogenesis has recently become a target of therapy for Type 2 diabetes. The following proposal describes experimental procedures by which to block the gluconeogenesis pathway and so prevent excessive glucose production, a key component of Type II diabetes.
Significance
Current diabetic medications focus on preventing insulin resistance or inefficiency, the key causative agents in the fight against Type II diabetes. These medications do not necessarily prevent excessive amounts of endogenous glucose from being generated through the gluconeogenesis pathway. The work described here will fulfill an unmet need in the fight to prevent Type II diabetes. The significance of this proposal is substantial and could have a positive impact for hundreds of millions of people around the globe who are diabetic. The end result of the research proposed here, in conjunction with currently used medications, may have a combinatorial positive effect on the health of diabetic and pre-diabetic patients by preventing an overexcess of glucose production.
Specific aims
The aims of this proposal are to develop a means by which to target the gluconeogenesis pathway to prevent type II diabetes. This can be accomplished by using inhibitors of enzymes involved in the gluconeogenesis pathway, such as fructose-1, 6 bisphosphatase, as well as the enzyme glycerophosphate dehydrogenase, located in the mitcochondia. Other compounds can be identified which act to block these or other enzymes involved in the gloconeogenesis pathway.
Materials and methods
The materials and methods will involve the use of known and potential new inhibitors of key enzymes involved in the gluconeogenesis pathway, such as the natural inhibitor AMP and related molecule for the enzyme fructose-1, 6-biphosphate (van Poelje et al., 2011). Variations of these molecules will be tested for their ability to inhibit enzyme synthesis in an in vitro assay that examines enzyme kinetics, with known substrates and products that can be quantified. Inhibitors that successfully block the activity of different enzymes involved in gluconeogenesis can then be tested in animal models. Rats, for example, can be administered the inhibitor and then their endogenous glucose levels can be examined. If their exogenous glucose levels go down without any adverse effects, then a clinical trial can be set up using healthy, nondiabetic human subjects. If these first phase clinical trials are successful in reducing endogenous levels of glucose in humans, then a second trial can be set up using actual diabetic patients. Successful completion of this trial will result in a potential new way to combat Type II diabetes through blocking gluconeogenesis, and will provide a more effective way to help people chronically afflicted with this disease.
In an analogous fashion, metformin has been used to inhibit the mitochondrial enzyme glycerophosphate dehydrogenase, and thus reduces the conversion of lactate and glycerol to glucose in the liver (Madiraju et al., 2014, Pernicova and Korbonits, 2014). As a result, metformin can successfully inhibit hepatic gluconeogenesis. It is therefore worthwhile to identify other compounds which can act in a manner that resemble metformin, so that an army of inhibitors can be used to block gluconeogenesis from taking place at different sites along the pathway. (Moneva et al., 2002).
Analytical section
These experiments could be initiated using modern biochemical methodologies to identify which compounds are able to efficiently inhibit a variety of enzymes that are involved in the gluconeogenesis pathway. Inhibitors can then be tested on animal models such as rats to determine whether they are effective in vivo, what dosage should be used, and to determine whether the inhibitors have any adverse or toxic effects. Those inhibitors that act to reduce endogenous glucose levels in animals can then be tested in humans. This general procedure can be used to seek out inhibitors at other stages of the gluconeogenesis pathway, with the concept that a cocktail of inhibitors would eventually be provided to patients to assist in the control of their diabetes.
References
American Diabetes Association; 2014, http://www.diabetes.org/diabetes-basics/type-2/
WebMD, 2014, Type II Diabetes; http://www.webmd.com/diabetes/guide/type-2-diabetes
Mayo Foundation for Medical Education and Research, 2014, Type II Diabetes ; http://www.mayoclinic.org/diseases-conditions/type-2-diabetes/basics/definition/con-20031902
van Poelje PD, Potter SC, Erion MD. Fructose-1, 6-bisphosphatase inhibitors for reducing excessive endogenous glucose production in type 2 diabetes. Handb Exp Pharmacol. 2011;(203):279-301. doi: 10.1007/978-3-642-17214-4_12.
Moneva MH, Dagogo-Jack S.Multiple drug targets in the management of type 2 diabetes. Curr Drug Targets. 2002 Jun;3(3):203-21.
Madiraju AK, Erion DM, Rahimi Y, Zhang XM, Braddock DT, Albright RA, Prigaro BJ, Wood JL, Bhanot S, MacDonald MJ, Jurczak MJ, Camporez JP, Lee HY, Cline GW, Samuel VT, Kibbey RG, Shulman GI. Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase. Nature. 2014 Jun 26;510(7506):542-6. doi: 10.1038/nature13270. Epub 2014 May 21.
Pernicova I, Korbonits M. Metformin--mode of action and clinical implications for diabetes and cancer. Nat Rev Endocrinol. 2014 Mar;10(3):143-56. doi: 10.1038/nrendo.2013.256. Epub 2014 Jan 7.
