Shi, Min (2019) The effects of supplementation with blueberries and yoghurt and their bioactive components on obesity related comorbidities. PhD thesis, Victoria University.

Abstract

Obesity is a leading global health problem contributing to various comorbidities, including cardiovascular disease, type II diabetes mellitus (T2DM) and hypertension. Current conventional medical interventions for the treatment of these diseases are limited with varied efficacy and associated side effects. Thus complementary dietary or alternative therapies have become increasingly popular as alternative treatment methods for obesity and its related comorbidities. There is an immense diversity of plant and animal products, which may be effective agents for attenuating the development of obesity, T2DM and hypertension as they often contain a vast array of bioactive compounds that have been associated with significantly positive health outcomes with minimal side effects. However, the effectiveness of many of these foods and their bioactive molecules in the treatment of human diseases has yet to be fully explored. The overall focus of this thesis was to determine the effectiveness of supplementation with blueberry, yoghurt and their important respective bioactive components, cyanidin-3-O-βglucoside (C3G) and peptides, alone or in combination, on the risk factors of obesity and its related comorbidities. To undertake the overall focus of this thesis, the bioactive peptides were extracted from yoghurt and then these peptides as well as C3G were utilised in a human primary skeletal muscle cell culture experiment. This work was further developed with experimentation with these compounds in addition to supplementation with blueberry and yoghurt in isolation and in combination in a high-fat-high-carbohydrate (HFHC) induced obese mouse model. In order to produce bioactive peptides with angiotensin converting enzyme (ACE) inhibitory activity from yoghurt, the optimal fermentation conditions were determined. The results showed that peptides with ACE inhibitory activity (1.47 ± 0.04 mg/mL of IC50 values) were those obtained from yoghurt fermented by 1% of L. helveticus with Flavourzyme® for 12 h. After further separation, one fraction of peptides showed the highest ACE inhibitory activity with an IC50 of 35.75 ± 5.48 μg/mL. The total content of anthocyanidins and C3G in thirteen brands of blueberry samples were also analysed in order to choose the suitable brand for animal experimentation. Based on these results, Bhatti & Manj blueberries were selected attributed to their higher content of total cyanidin. In order to determine the effects of C3G and peptides on the regulation of glucose metabolism, different concentrations of C3G and peptides, alone or in combination, on glucose uptake and mRNA expression of key genes were investigated in human primary skeletal muscle cells derived from obese and obese diabetic (obese T2DM) individuals. The results showed that both low and high concentration of peptides and the combination of these peptides with high C3G concentration significantly enhanced glucose uptake in the presence or absence of insulin in the human primary skeletal muscle cells derived from obese individuals. However, high peptide concentration only increased glucose uptake in the absence of insulin in the obese T2DM group. In the obese group, high concentration of peptide alone and its combination with low C3G down-regulated the mRNA expression of angiotensin II receptor, type 1 (AGTR-1), and up-regulated the mRNA expression of insulin receptor substrate 1 (IRS-1), and glucose transporter 4 (GLUT4). In the obese T2DM group, the expression of AGTR-1 was decreased at high peptide concentration and its combinations with C3G. To further determine whether C3G and peptides, and their original food sources, blueberries and yoghurt, could attenuate obesity and its related comorbidities, a 16-week animal study using HFHC diet induced obese male C57BL/6 mice was conducted. It was found that blueberry and yoghurt alone, and the combination of peptides and C3G significantly reduced both systolic and diastolic blood pressure. Moreover, yoghurt significantly reduced body weight, percentage body fat and improved intraperitoneal glucose tolerance compared to the HFHC (control) group. Furthermore, peptides and its combination with C3G resulted in a significant reduction in percentage body fat and improvement of intraperitoneal glucose tolerance. However, energy expenditure and locomotor behaviour did not alter in any treatment groups compared to the HFHC group. The mRNA expression of multiple genes related to glucose metabolism in the skeletal muscle (extensor digitorum longus (EDL) and soleus) of mice supplemented as detailed above was determined by quantitative ’real-time’ polymerase chain reaction (PCR). In EDL, yoghurt alone up-regulated the expression of AMPK, IRS-1, PI3K and GLUT4, and down-regulated the expression of AGTR-1. The combination of blueberry and yoghurt down-regulated the mRNA expression of AGTR-1 and FoxO1 in EDL. Furthermore, the combination of C3G and peptides also down-regulated the mRNA expression of AGTR-1 and up-regulated the mRNA expression of GLUT4 in EDL. In soleus, blueberry and yoghurt supplementation alone and their combination down-regulated the mRNA expression of AGTR-1, and upregulated the mRNA expression of GLUT4. Therefore, the outcomes of this thesis highlight that yoghurt and its peptides have the potential to reverse or attenuate metabolic disturbances associated with developing obesity, diabetes and hypertension. Blueberries reduced blood pressure while no inhibition on body weight and body fat gain was observed. Furthermore, C3G may not be effective in eliciting beneficial effects on obesity, diabetes and hypertension possibly due to the low dosage utilised. In conclusion, the results presented within the current thesis support that yoghurt as a fermented dairy product may be a beneficial additive to functional foods or utilized as a dietary component. Blueberry plays a significant role in the treatment of hypertension, which represents a potentially promising dietary intervention worthy of further investigation.

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