EFFECTS OF SOME FLAVONOIDS ON OBESITY-LINKED TYPE 2 DIABETES: ROLE OF AUTOPHAGY

Abstract

Diabetes Mellitus (DM) is a group of metabolic disorders characterized by an increased level of blood glucose due to defects in insulin secretion or action or both. Among all diabetic conditions, type 2 diabetes is most prevalent and accounts for nearly 90-95% of patients diagnosed with diabetes worldwide. Type 2 diabetes mellitus (T2DM) is a metabolic disease characterized by elevated blood glucose level and insulin resistance. The world health organization (WHO) reported that the worldwide number of people with diabetes has risen to 422 million in 2014 from 108 million in 1980 with an estimated 1.5 million deaths in 2012 (World Health Organization, 2016). As the knowledge of heterogeneity of this disorder increases, there is a need to search for more efficacious agents with lesser side effects. Although the modern medicines and therapies can mitigate diabetes to some extent, there are several unprecedented complications that need to be addressed through a holistic approach of therapy. Towards this end, a validation of our traditional knowledge of Ayurveda and then combining it with the modern scientific approach may provide the best possible solution to it. The traditional plant-based medicines are safe, effective, economical, non-toxic with no or lesser side effects as compared to other synthetic drugs. Hence these herbal plants or active phytoconstituents are considered to be the best possible alternative candidates in diabetes management. The present thesis entitled “Effects of some flavonoids on obesity-linked type 2 diabetes: role of autophagy” deals with evaluating the anti-obesity and antidiabetic effects of some flavonoids using in vitro and in vivo approaches followed by evaluating the autophagy stimulatory activity of these flavonoids. Out of four flavonoids selected for this study, kaempferol was found to be most potent in stimulating autophagy and restoring β-cell mass and function. Thus, kaempferol was chosen for further detailed and mechanistic studies using in vitro, ex vivo and in vivo models to establish the exact cross-talks among various pathways in causing autophagy and its related antidiabetic effects. In the beginning, the Chapter 1 introduces briefly the present scenario of diabetes and therapeutic drugs available for the management of this metabolic disorder. It also deals with the key mechanisms and major signaling pathways involved in the pathophysiology of this disease that needs to be targeted for its prevention and cure. These mechanisms can serve as a Abstract ii major target to select a natural molecule for its respective efficacy. Finally, the detailed objective to be attained in this study is specified in this chapter. Followed by this, Chapter 2 presents: (i) a detailed review on diabetes and its link to obesity; (ii) detailed mechanisms involved in pathophysiology of diabetes with special emphasis on insulin resistance and β-cell failure; (iii) conventional and current therapies for diabetes management and their limitations; (iv) understanding the molecular mechanism of autophagy and its role in obesity-linked diabetes, insulin resistance and β-cell failure with major focus on molecular pathways and their therapeutic targets; (v) recent research detailing benefits of phytochemicals and their characteristic to target multiple pathway on various target tissues and (vi) the hypothetic idea behind the present thesis to elucidate the efficacy of flavonoids for their potential to stimulate autophagy and their antidiabetic role. Each of these hypotheses was further explored in subsequent chapters of the thesis. The main objective of the present work is to decipher anti-obesity, antidiabetic and autophagy stimulatory effects of flavonoids followed by understanding their mode of action. There are various in vitro and in vivo assays which are already established to confirm a compound to be anti-obese and antidiabetic. All these parameters are elaborated in Chapter 3 of this thesis. In this regards, this chapter contains the details about various cell lines, ex vivo and in vivo models used in the study and the overall principles and methodology of all the experimental assays that were performed to identify the autophagy stimulatory and antidiabetic effects of flavonoids. Hence, various biochemical methods such as cell proliferation assays, adipocyte differentiation assays, Oil Red O staining, glucose uptake assay, specific assays to detect apoptosis and autophagy, which were used initially to screen the selected flavonoids’ potential as antidiabetic or anti-obese potentiality are described in detail. Further, the principles of the biological mechanisms were studied at the transcriptional and translational levels of various genes to evaluate their mode of actions intracellularly. T2DM is a metabolic disorder with an elevated blood glucose level and insulin resistance, are mostly associated with obesity thus indicating a direct correlation between these two pathological conditions. Recent studies showed that dysfunctional autophagy is involved in the loss of β-cell mass and function, hence, demonstrating pathophysiology of obesitylinked diabetes. Therefore, identifying agents that could restore autophagy in degenerated β- cells are of great importance under the current therapeutic regime. Currently, an active area of diabetes research focuses on identifying naturally occurring phytochemicals that can be Abstract iii modulated or developed into therapeutic for diabetes due to their intriguing role in treating various diseases. One such group of phytochemicals found extensively in various plants is flavonoid. In Chapter 4, few of these flavonoids were screened using in vitro and in vivo models for their anti-obesity and antidiabetic effects. These flavonoids were further analyzed for their potential to stimulate autophagy under lipid overload conditions. Our results showed that among all-selected flavonoids, kaempferol had potent antidiabetic and autophagy stimulating activities as obtained from initial screening tests and thus selected for further detailed and mechanistic studies. Lipotoxicity of pancreatic β-cells is the pathological manifestation of obesity-linked type II diabetes. Autophagy is a lysosomal degradation pathway of dysfunctional macromolecules and organelles which protects cells from stressed conditions and acts as an adaptive prosurvival response. In Chapter 5, we intended to determine the cytoprotective effect of kaempferol on pancreatic β-cells undergoing apoptosis in palmitic acid (PA)-stressed condition. The data showed that kaempferol treatment increased cell viability and antiapoptotic activity in PA-stressed clonal pancreatic β-cells (RIN-5F cell line) and isolated primary cultures of rat islets. Further, it was found that kaempferol exerts its cytoprotective actions by inducing autophagy via AMPK/mTOR signaling pathway in PA-stressed pancreatic β-cells. Chapter 6, depicts the molecular mechanism involved in cross-talk between kaempferolmediated autophagy and its anti-apoptotic effect in PA-stressed β-cells. Endoplasmic reticulum (ER) stress generated due to the accumulation of lipid in β-cells is one of the major features of pathological conditions involved in cell death under lipid overload conditions. Interestingly, autophagy is involved in the alleviation of lipid-induced ER stress thus protecting β-cell from apoptosis. On the other hand, autophagy is also reported to degrade lipid droplets in hepatocytes, however, its role in lipid metabolism of β-cell is rarely reported. Based on this information in this study, the inhibitory effects of kaempferol-induced autophagy on PAinduced lipid deposition and ER stress were evaluated. Our studies showed that kaempferolinduced AMPK/mTOR-mediated autophagy abolished the PA-induced ER stress and lipid accumulation which seems to be one of the possible mechanisms involved in the kaempferolmediated restoration of β-cell mass. Additionally, this study revealed that kaempferol-induced autophagy also restored the β-cell function. Abstract iv In Chapter 7, we intended to validate our findings in a more promising diabetic model, the high fat diet (HFD) and streptozotocin (STZ)-induced type 2 diabetic mice, an alternative animal model of type 2 diabetes, simulating human syndrome which has been extensively used to test antidiabetic effects of various drugs/compounds. Our results exhibited that kaempferol treatment resulted in significant improvement in glycemic control and metabolic profile in HFD-STZ-induced diabetic mice. The improved glucose homeostasis was found to be associated with increased insulin sensitivity. Further, it was evident that kaempferol-induced autophagy is involved in the protection of β-cell mass and function which in part ameliorates diabetes in HFD-STZ induced diabetic mice. The two pathophysiologic abnormalities which link obesity to T2DM, are β-cell failure and insulin resistance. Hence, in Chapter 8, the effects of kaempferol on hepatic insulin resistance and insulin signal transduction were evaluated in vitro and in vivo in PA-challenged HepG2 cells and livers of HFD-STZ-induced diabetic mice respectively. Dysfunctional autophagy and ER stress due to increased ectopic lipid accumulation in hepatocytes are the pathological manifestation involved in the lipid-induced impairment of insulin signaling and thus insulin resistance. In this study, lipid inhibitory effects of kaempferol were determined in in vitro and in vivo models. Further, to explore the possible underlying mechanism, the role of autophagy and β-oxidation were investigated. Our results showed that kaempferol exerted its lipid inhibitory effects in hepatocytes through autophagy and β-oxidation mechanism under lipid overload conditions. Further, kaempferol treatment significantly abolished the HFDmediated increase in ER-stress induced JNK (Thr183/Tyr185) and IRS-1 phosphorylation (Ser307) which finally helped in the restoration of insulin-stimulated phosphorylation of Akt (Ser473). Together, this study revealed that kaempferol-induced AMPK-mTOR-mediated autophagy is involved in the alleviation of hepatic lipid accumulation and impaired insulin signal transduction in PA-induced HepG2 cells and livers of HFD-STZ-induced diabetic mice which, in part, ameliorates insulin resistance and hyperglycemia. Finally, Chapter 9 summarizes the major findings of the current thesis and provides suggestions for future work in this area. The scientific findings dealt with in this thesis may be of use to the future researchers working in this area. Last but not least, Chapter 10 of this thesis listed the bibliography which was consulted in course of the present work.