Thunuguntla, Venkata Bala Sai Chaitanya (2022) Improved satiety signalling in obesity with a novel combination treatment of Caralluma fimbriata Extract and Lorcaserin. PhD thesis, Victoria University.

Abstract

Being overweight and obese are risk factors for multiple chronic diseases, including hypertension, type II diabetes mellitus, and cardiovascular diseases. These factors drive the development of new treatments to control obesity. The regulation of food appetite is a critical element of energy balance in obese people. Hypothalamic neurons of the brain region play a crucial role in balancing hunger and satiety for an individual. The hypothalamus's arcuate nucleus (ARC) contains Proopiomelanocortin (POMC) neurons that mediate food satiety signalling. They express one of the serotonin receptors called 5HT2cR, which binds to the ligand serotonin to activate satiety signalling through the anorexigenic pathway. Hence, the 5HT2cR is proposed as one of the potential targets for regulating obesity and overeating disorders. Several agonists have been developed to increase the functional activity of 5HT2cR and further enhance satiety signalling. This satiety signalling is dysregulated in overeating diseases like Prader-Willi syndrome and Bardet–Biedl syndrome. Studies have shown that a succulent plant, Caralluma fimbriata extract (CFE), can reduce body weight and waist circumference. In addition, preliminary data on modified fibroblasts (3T3 cells) showed that CFE increased expression of the full-length 5HT2cR mRNA compared to the truncated/nonfunctional variant. However, the mechanism through which CFE reduces obesity remains undefined. The FDA approved the 5HT2cR-specific agonist Lorcaserin to treat obesity in 2012. However, the drug Lorcaserin has limited use for the long-term treatment of obesity due to concerns about side effects and it was withdrawn from sale in the USA in 2020. The current study aims to investigate whether the combination of CFE and a lower dose of lorcaserin can increase 5HT2cR activation and reduce diet-induced obesity. The CFE is predicted to increase 5HT2cR expression, thereby increasing satiety signalling when activated by the specific agonist lorcaserin. The study comprises three phases; the first phase involved human SHSY5Y neuroblast cell culture as a model to study 5HT2cR expression studies in response to treatment with CFE. The treatment was carried out for 48h and 96h duration with different concentrations of CFE. In addition, these SHSY5Y cells can be differentiated from fibroblasts to mature neurons. These differentiated neurons retain the property of expressing 5HT2cR. Therefore, these neurons were treated with CFE for 48h and 96h with different concentrations (ranging from 0.1 to 50 µg /mL) of CFE. The treated cells were analysed for differential gene expression studies, including full-length (functional) and truncated 5HT2cR expression by quantitative PCR and pilot-scale next-generation sequencing (NGS) for further metabolic analysis. The results showed a significant two-fold increase in the expression level of the 5HT2cR functional receptor in CFE-treated neurons at 25 µg /mL (1.87-fold at 48h and 2.41-fold at 96h of CFE treatments with p value 0.01 and 0.03 respectively) and at 50 µg /mL (2.84-fold at 48h and 2.54-fold at 96h of treatments with p values <0.001 and 0.02 respectively) concentrations, respectively. These results encouraged follow-up of the changes in metabolic pathways in the presence of CFE-treated neurons. The whole transcriptome analysis revealed anorexigenic genes like PCSK1 and BDNF, and 5HT2cR were upregulated. In contrast, orexigenic genes like NPY and NPY2R were down regulated. In addition, the longer (96h) CFE treatment showed a greater increase in functional 5HT2cR expression in qPCR and transcriptome analysis. The second phase investigated the effect of combined CFE and Lorcaserin treatment in a highfat diet (HFD) -induced obese mouse model. The study measured physiological parameters, including food and water intake and body weight changes. Since 5HT2cR activation also influences mood and behaviour, the current study included observations of behavioural changes in anxiety, depression, and exploration parameters. The six-week-old mice were housed at standard facility conditions and fed a HFD for eight weeks to induce obesity. The animal groups were divided as control diet, HFD, HFD with CFE (100mg/kg/day) treatment, HFD with Lorcaserin (5mg/kg/day), and HFD with CFE and Lorcaserin (combination: CFE+LOR) for eight weeks of treatment. The animals were measured weekly for food and water intake and body weight. The treatments of hydroethanolic extract of CFE (100mg/kg body weight (bwt) /day) and Lorcaserin (5mg/kg bwt/day) were mixed in a jelly form and given at a weight below 10% of the daily food intake of the mice. In the CFE+LOR-treated group of mice, the results showed a reduction of anxiety and depression parameters to the normal range compared to the HFD group. The explorative behaviour in the y-maze test didn't show significant variation in any parameters. The food intake was trended down in CFE and CFE+LOR groups compared to the HFD group; however, the data was not significant. On the other hand, the HFD group significantly decreased water intake compared to CFE+LOR treated group. The animal study also measured energy expenditure by indirect calorimetry and body fat composition with EchoMRI for all the groups. The analysis revealed fat content decreased significantly in CFE and CFE+LOR treatment groups compared to the HFD group. The Promethion indirect calorimetry and locomotor activity system was used for three days to determine energy expenditure. There was a significant increase in the overall energy expenditure of treated groups compared to HFD. The sleep cycle and daily walking percentage also improved significantly in CFE+LOR-treated mice compared to HFD and LOR-treated mice. Overall, CFE and CFE+LOR treatments protected the animals against HFD exposure and reduced body weight gain and fat deposition. The water intake during the dark cycle was significantly more in CFE and CFE+LOR groups than in HFD mice. Even though the CFE treatment group showed reduced weight gain and fat deposition, the CFE+LOR group showed better resistance to HFD. On the other hand, the LOR treatment alone did not significantly alter weight gain. In addition, obesity-related comorbidities include an increased risk of vascular endothelial dysfunction. Functional analysis of the abdominal aortic rings showed that all the treatments improved the vasodilatory response in HFD-fed mice. In conclusion, the study used a novel combination therapy consisting of CFE and a relatively low dose of Lorcaserin to reduce food appetite and obesity. The CFE was able to increase the functional 5HT2cR expression in SHSY5Y-neurons. In HFD-induced mice, CFE and CFE+LOR treatments showed reduced food intake compared to the HFD group. In addition, the behavioural data showed reduced anxiety and depression parameters in the presence of CFE+LOR treatments compared to individual treatments of CFE or LOR alone. The study summarises that CFE alone and CFE with a low dose of Lorcaserin can reduce obesity and improve parameters of mood and vascular function in an HFD-induced model of obesity. Further research is required to confirm if this combination approach will be a potential therapy for obesity and overeating diseases with a lowered risk of possible side effects compared to Lorcaserin alone.

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