Treatments for Obesity — a Background of Current Therapies and Novel Approaches
Obesity
Obesity is often defined as the imbalance in the ratio of fat accumulation to energy expenditure generally with a Body Mass Index (BMI) over 30. It serves to be a major risk factor for many other diseases including cardiovascular disease, stroke, diabetes, musculoskeletal disorders such as osteoarthritis, and many varieties of cancer. The causes of obesity are preventable through reformations in diet—as the fundamental cause of obesity is intaking more calories than used; however, no country has yet to reverse this epidemic, indicating that exploring the presence and impact of other factors may be beneficial. Such factors include genetics, disruption of sleep, stress and fluctuations in emotions, lack of resources, socioeconomic status, medications, health conditions, and the imbalance of hormones, the focus of this study.
Leptin
Leptin is a hormone released by adipose tissue that helps maintain a normal body weight through the regulation of hunger and satiety levels. When fat stores are created after eating, they secrete leptin which travels through the bloodstream, passes through the blood-brain barrier, and binds to cytokine-1 receptors in the hypothalamus. This negative feedback loop, mediated by the levels of leptin, is able to successfully suppress one’s appetite and sustain a normal body weight. Moreover, it’s important to note that most people obtain roughly the same number of fat cells as we were born with; what differs between an individual with obesity is the size of the adipose cells and the number of triglycerides stored in them. Likewise, another function of leptin is to partake in the reduction of triglycerides by inhibiting fat synthesis and stimulating the breakdown of fat through beta-oxidation, a catabolic pathway in which carbons are removed from fatty acids to generate acetyl-CoA and ATP.
At a molecular level, leptin typically affects change through the JAK-STAT signaling pathway. The Leptin Receptor (LepR) is a transmembrane cytokine I receptor that functions similarly to a tyrosine kinase. When leptin binds to LepR, the receptors dimerize, and the janase-activating kinases (JAK) are located on the intracellular side of LepR phosphorylate each other and the tyrosine forming a docking site for the Signal Transducers and Activators of Transcription (STAT). The STAT proteins, often STAT3, bind onto the activated tyrosine, delocalize from LepR, and dimerize, and enter the nucleus where they bind to DNA to control gene expression. For example, STAT3 often upregulates neuropeptides, like arcuate pro-opiomelanocortin (POMC), which play major roles in satiety and metabolism. The more leptin that binds to regions of the arcuate nucleus, the less hungry one should feel (in the absence of environmental and neurological factors). As a result of this binding, the arcuate nucleus triggers the sympathetic nerve to secrete norepinephrine which associates with B3 Adrenergic receptors leading to a host of biosignaling events, including the increase in the second messenger cAMP. Protein Kinase A is activated by cAMP which leads to the increased expression of the UCP1 gene, encoding for thermogenin, also known as the uncoupling protein, which expedited beta-oxidation by causing the energy from the fatty acids to be dissipated as heat. In short, the effects of leptin result from its binding to LepR.
Due to leptin’s anorexic effects, it may seem as though obese individuals will lack sufficient leptin. However, this is often not the case—most cases of obesity are associated with hyperleptinemia, excessive circulating levels of leptin, rather than hyperleptinemia, abnormally low levels of leptin (although both conditions are linked with obesity). This counterintuitive phenomenon can be attributed to leptin resistance.
In individuals with excess fat stores, high amounts of leptin are secreted to suppress appetite; however, the brain stops responding to this leptin. This is due to inflammation-related defects in the leptin receptor often caused by excess glucose intake. Without functional LepR for leptin to bind to, the signaling pathways fail to occur thus leading the body to detect lower rates of leptin despite the high number of fat stores. Consequently, the hypothalamus’ insensitivity to leptin and disruptions in the JAK-STAT signaling pathway leads to unregulated hunger and decreased fat metabolism. Hunger will persist causing an individual to consume more calories, break down fewer triglycerides, and store more fat, hence increasing the rate of circulating leptin levels and further exacerbating this process. In fact, this resistance may become so severe that the body turns to a “starvation” mode contributing to excess binging of food.
Leptin Neutralizing Antibody and Control Antibody
The leptin antibody was developed by first separating the parental antibody from a phage display human single chain variable fragment (ScFv) antibody library, containing recombinant proteins of variable light or variable heavy chains. The phage display was able to study the interactions between proteins through the genetic engineering of bacteriophages. Genes were inserted into bacteriophages leading them to express the corresponding protein on the surface. Multiple iterations of this method with many different inserted genes led to the creation of the phage library. The bacteriophages were then exposed to select targets to determine their binding capabilities. Unbound bacteriophages were washed while bound bacteriophages were eluted through true affinity chromatography. Eluted bacteriophages infect new host cells allowing for amplification and repeated multiple times to increase affinity between the expressed proteins. This resulted in the identification of the parental leptin-neutralizing antibodies.
Because the antibody was identified from a human phage library, injection into mice may elicit an immune response. Consequently, LepAB was “mouserized” using the KABAT/IMGT CDR-grafting technique. In the KABAT numbering system, amino acid residues were numbered based on their variable region, statistics were taken based on the amino acid frequency in a position, and three hypervariability regions with highly conserved residues clustered at one side of the folded domain for antigen recognition. In IMGT, all protein sequences from the IG family are based on the amino acid sequence alignment of germline V genes. These two methods define complementary determining regions, framework residues from light and heavy chains that affect binding affinity and specificity of antibody-antibody interaction resulting in optimal performance. Then, to prevent immune responses to the leptin-neutralizing antibody while retaining the human framework, CDR grafting was employed to reshape the antibody. IgBLAST and IMGT/V-Quest were used to identify complementary determining regions that would mediate antibody-antibody interactions; the variable heavy (VH) and variable light (VL) sequences of the parental LepAB were compared to a mouse germline sequence database in order to produce the template. Finally, the rest of the antibody was synthesized by cloning the VH and VL sequences into the IgG1 mouse antibody and the light chain backbone. Mouse IgG1 is an isotype control, a type of antibody that lacks target specificity but matches the class and type (immunoglobulin G) of the primary antibody. Accordingly, the IgG1 antibody was used as the control.
In short, the KABAT/IMGT numbering systems identify complementary determining regions essential to the antibody’s interaction. These highly conserved sequences are then grafted onto similar VH and VL antibody sequences in the mouse, identified using BLAST, and then integrated into the mouse IgG1 antibody control.
Liraglutide
Liraglutide, commonly known as Saxenda, is a diabetic medication with secondary effects of weight reduction. Liraglutide is a Glucagon-like peptide 1 (GLP1) agonist, as it is 97% homologous GLP-1, thus exhibits a similar mechanism of action without facing the consequence of rapid degradation by other regulatory enzymes, such as DPP-4. After intaking food or accumulating nutrients in the intestinal lumen, the gut secretes incretins, one of which being GLP-1. GLP-1 then binds to its receptor on the beta cells of the pancreas, activating adenylyl cyclase which converts ATP to cyclic AMP (cAMP). cAMP then triggers the release of insulin, leading to a decrease in blood glucose. Similarly, by inhibiting high voltage-activated calcium channels associated with glucagon exocytosis, GLP-1 and GLP-1 agonists are able to suppress the activation of the alpha cell of the pancreas and lower glucagon secretion. GLP-1 also delays gastric emptying causing the GI tract to be stretched and distended for a longer period of time, and enhances the stimulation of the vagus nerve as GLP-1 binds to the chemoreceptors and activate vagal afferent signals that trigger the Nucleus Tractus Solitarii (NTS) in the hypothalamus, inducing satiety.
