Elucidation of molecular events underlying hypoxia induced dysfunctions in 3T3-L1 adipocytes and possible amelioration with Bilobalide and Curcumin

Abstract

Obesity and related complications have increased substantially over the past few decades. It is a prominent risk factor for insulin resistance, hypertension, type 2 diabetes and cancer. Obesity is characterised by excessive expansion of adipocyte size and adipose tissue mass. This enlargement of adipocyte size (140-180 µm) exceeds the normal oxygen diffusion distance (100 µm) and compromises the effective oxygen supply, which lead to local hypoxia. The response to low O2 levels is accomplished through the activation of specific transcription factor, hypoxia inducible factor 1 (HIF-1). The HIF-1α subunit of this heterodimeric transcription factor is considered as the molecular oxygen sensor. When cellular O2 levels are sufficient, this protein is continuously synthesized but is immediately targeted for proteasome degradation. The low O2 level induces stabilization, nuclear translocation, and activation of this transcription factor. HIF-1α expression is directly linked to adiposity and is decreased following weight loss. So the hypoxia-signaling pathway is expected to provide a new target for the treatment of obesity-associated complications. Inhibition or downregulation of the HIF-1 pathway could be an effective target for the treatment of obesity related hypoxia. In this study, we evaluated the effect of hypoxia on functions of 3T3-L1 adipocytes emphasising on oxidative stress, endoplasmic reticulum (ER) stress, mitochondrial dysfunctions, inflammation, and insulin resistance. We also evaluated the protective role of two phytochemicals, bilobalide and curcumin, on hypoxia induced alterations. Hypoxia was induced in differentiated 3T3-L1 adipocytes on 9th day by incubating in hypoxic chamber at an atmosphere of 1% O2, 94% N2, 5% CO2, and at 37o C for 24 hrs. The control cells were incubated in an atmosphere of 21% O2 and 5% CO2 at 37o C. The cells were treated with different concentrations of bilobalide (10 µM, 20 µM & 50 µM) and curcumin (5 µM, 10 µM & 20 µM) during hypoxic period (24hrs). Acriflavine (5 µM) is used as positive control which is an HIF-1α inhibitor. The thesis is divided into 5 chapters including summary and conclusion. A general introduction dealing with obesity and its prevalence, obesity related metabolic complications, importance of adipose tissue in obesity, adipose tissue hypoxia, the effect of hypoxia on key functions of adipocytes, and the role of natural products in obesity related xx complications, a brief note on the pharmacological properties of bilobalide and curcumin etc., along with aims and objectives of the study have been described in chapter 1. The chapter 2 highlights how hypoxia affects the physiological functions of 3T3- L1 adipocytes, emphasizing on HIF-1α expression, lactate release, oxidative stress and ER stress and possible protection with bilobalide and curcumin. The results revealed that hypoxia significantly altered all the vital parameters of adipocyte biology. There was a significant increase in HIF-1α expression, lactate release, ROS production, lipid and protein oxidation, in hypoxia treated groups compared with normoxic group. In addition, a reduction in antioxidant enzymes status was observed in hypoxic group. The expression of Nrf2/HO-1 and ER stress markers (GRP78, ERO1-Lα, PDI, PERK, IRE-1α, ATF-6 & CHOP) were significantly upregulated in hypoxic groups compared to normoxia. Bilobalide and curcumin attenuated the expression of HIF-1α, the hypoxic marker and protected 3T3-L1 adipocytes from hypoxia induced oxidative stress and ER stress. Chapter 3 depicts hypoxia induced mitochondrial dysfunctions in 3T3-L1 adipocytes and its protection by bilobalide and curcumin. Various parameters relevant to mitochondrial functions like superoxide production, aconitase activity, transmembrane potential, integrity of mitochondrial permeability transition pore, oxygen consumption rate and ATP content, proteins involved in oxidative phosphorylation, expression of genes involved in mitochondrial biogenesis and proteins involved in mitochondrial structural dynamics were analysed in all the normoxic and hypoxic groups. Hypoxia impaired all the vital parameters relevant to mitochondrial functions in differentiated 3T3-L1 cells. Bilobalide and curcumin protected 3T3-L1 adipocytes from adverse effects of hypoxia by enhancing mitochondrial biogenesis, mitochondrial functional performances and by controlling mitochondrial dynamics via downregulating HIF-1α expression. Chapter 4 explains the crosstalk between hypoxia induced inflammation, and insulin resistance and, also secretion of proangiogenic factors in 3T3-L1 adipocytes and possible reversal with bilobalide and curcumin. Hypoxia significantly increased the release of TNF-α, IL-6, IL-10, MCP-1 and IFN-γ, leptin, and resistin, the adipokines that induce inflammation and insulin resistance in adipocytes. But the secretion of adiponectin, a beneficial antidiabetic adipokine was significantly reduced in hypoxia treated adipocytes. Hypoxia also showed an increased mRNA expression of TLR4, the receptors of free fatty acids. Enhanced TLR4 activation in combination with increased glycerol release activated xxi inflammatory pathways, NF-κB and JNK signaling in hypoxic groups. Activation of NF- κB and JNK signaling pathways by hypoxia and subsequent higher expression of cytokines impair insulin signaling cascade by mediating serine phosphorylation of IRS-1 and by downregulating the expression of IRS-2. However, we observed an increased basal glucose uptake in hypoxia, in response to increased GLUT1 expression. But there were no significant changes in expression of GLUT4 after 24hrs of hypoxia. Bilobalide and curcumin ameliorated hypoxia-induced inflammation in 3T3-L1 adipocytes and improved insulin signaling. Hypoxia also increased the release proangiogenic factors (MMP-2, MMP-9, VEGF, angiopoietin like protein 4) in 3T3-L1 adipocytes. Bilobalide and curcumin significantly reduced the expression of proangiogenic factors via reducing hypoxia and inflammation. Chapter 5 describes the overall summary and conclusion of the study. Based on these results we presume that HIF-1α represent a viable therapeutic target for the treatment of obesity related hypoxia.