INTRODUCTION
Metformin is the most widely prescribed drug for the treatment of type II diabetes. This drug is administered for the purpose of lowering blood glucose; yet, the mechanism of this effect remains unclear (Baur & Birnbaum, 2014). Metformin is generally known to inhibit gluconeogenesis (Hundal et al., 2000; Lee et al., 2013; Stumvoll et al., 1995), a pathway that creates glucose out of building blocks amassed from the liver. Through this, blood glucose levels rise, contributing to hyperglycemia in type II diabetic patients (National Institute of Diabetes and Digestive and Kidney Diseases [NIDDK], 2016). Several theories exist regarding the mechanism by which metformin works (Lee et al., 2013; He & Wondisford, 2015; Mráček, Drahota & Houštěk, 2012; Madiraju et. al, 2014), but a definitive mechanism remains to be identified. This study hypothesizes that metformin inhibits gluconeogenesis through the inhibition of pyruvate carboxylase (PC), an enzyme involved in the first step of gluconeogenesis. This study will observe the potential effect of metformin in this initial step of the gluconeogenic pathway.
During gluconeogenesis, lactate is converted into oxaloacetate, which is later reduced to form malate (Kiran, 2015). This initial conversion of lactate into oxaloacetate is catalyzed by PC, and NADH is a coenzyme necessary for the later conversion of oxaloacetate into malate, catalyzed by malate dehydrogenase (MDH). Using a diode array spectrophotometer, NADH concentrations will be measured over time under varying concentrations of metformin. Data will be used to describe the rate of PC catalysis on gluconeogenesis, helping to indicate the effect of metformin on catalysis of this reaction. Furthermore, 13C-NMRs will be performed in order to assess the functional groups of pyruvate, oxaloacetate, and biotin, a prosthetic group of PC which interacts with PC and enables it to function (Chaambra, 2015). These substances were then each mixed with metformin, and the 13C-NMR of the mixture was compared with the individual 13C-NMRs for metformin and the appropriate substance to ascertain changes to the molecules once combined.
During gluconeogenesis, lactate is converted into oxaloacetate, which is later reduced to form malate (Kiran, 2015). This initial conversion of lactate into oxaloacetate is catalyzed by PC, and NADH is a coenzyme necessary for the later conversion of oxaloacetate into malate, catalyzed by malate dehydrogenase (MDH). Using a diode array spectrophotometer, NADH concentrations will be measured over time under varying concentrations of metformin. Data will be used to describe the rate of PC catalysis on gluconeogenesis, helping to indicate the effect of metformin on catalysis of this reaction. Furthermore, 13C-NMRs will be performed in order to assess the functional groups of pyruvate, oxaloacetate, and biotin, a prosthetic group of PC which interacts with PC and enables it to function (Chaambra, 2015). These substances were then each mixed with metformin, and the 13C-NMR of the mixture was compared with the individual 13C-NMRs for metformin and the appropriate substance to ascertain changes to the molecules once combined.
The purpose of this study is to observe the effects of metformin on PC catalysis during gluconeogenesis. This study hypothesized that metformin in concentrations ranging from 250µM - 500µM inhibits gluconeogenesis through inhibition of pyruvate carboxylase (PC), as well as that pyruvate, oxaloacetate, and biotin will each lose a carbonyl group and gain an amine group once mixed with metformin. Understanding of the metformin mechanism of action will allow for greater understanding of type II diabetes itself, as well as pave the way for improved treatment of this disease and even a potential cure, as well as will allow for better understanding and mitigation of metformin side effects, such as lactic acidosis, a debilitating and often fatal condition which is associated with metformin consumption in rare cases (Duong et al., 2013). Furthermore, observation of the effect of metformin on PC and general understanding of the regulatory role of PC on the gluconeogenesis pathway will help amass support for the metabolic control theory, which posits that major metabolic pathways like gluconeogenesis are not simply controlled by one primary enzyme, such as phosphoenolpyruvate carboxylase (PEPCK) in the case of gluconeogenesis, but rather are controlled by several enzymes and molecules working in tandem with one another in response to cellular stresses and environmental changes (Fell, 1997).
The implications of this research extend beyond purely academic advancement. Type II diabetes is on the rise, and is becoming more prevalent among younger demographics (CDC, 2017). It is of utmost importance to address this disease and to understand the mechanisms by which current treatments work to lower blood glucose levels, such that greater understanding of type II diabetes may be gained as well as novel treatments for this disorder may be developed. Type II diabetes can affect any person of any age, but is most likely to affect obese or overweight adults, and most commonly afflicts black, Native American, and Hispanic communities (Spanakis & Golden, 2014). These three ethnicities face the highest rates of poverty in the United States of America (Macartney, Bishaw & Fontenot, 2013), and it is well-documented that health outcomes are poorer among lower socioeconomic levels. Diabetes is a public health crisis which disproportionately affects communities that face internal and external pressures such that health outcomes and general quality of life are already reduced compared to the national average. Understanding the mechanism of metformin action will not only add to the growing body of knowledge regarding type II diabetes, but has human implications as well, and will add to the growing body of hope and optimism in afflicted communities across the nation as well as globally.
The implications of this research extend beyond purely academic advancement. Type II diabetes is on the rise, and is becoming more prevalent among younger demographics (CDC, 2017). It is of utmost importance to address this disease and to understand the mechanisms by which current treatments work to lower blood glucose levels, such that greater understanding of type II diabetes may be gained as well as novel treatments for this disorder may be developed. Type II diabetes can affect any person of any age, but is most likely to affect obese or overweight adults, and most commonly afflicts black, Native American, and Hispanic communities (Spanakis & Golden, 2014). These three ethnicities face the highest rates of poverty in the United States of America (Macartney, Bishaw & Fontenot, 2013), and it is well-documented that health outcomes are poorer among lower socioeconomic levels. Diabetes is a public health crisis which disproportionately affects communities that face internal and external pressures such that health outcomes and general quality of life are already reduced compared to the national average. Understanding the mechanism of metformin action will not only add to the growing body of knowledge regarding type II diabetes, but has human implications as well, and will add to the growing body of hope and optimism in afflicted communities across the nation as well as globally.