AbstractThe prevalence of overweight and obese individuals has been recognized globally for decades. Of all countries, United States has the highest rate of obesity. One third of its adult population is obese, and around two thirds are overweight. Moreover, childhood obesity has rapidly increased with 17% of children ages 2-19 are now obese. The American Medical Association (the nation’s leading physician’s organization) recently declared that obesity is a “disease”. Obesity increases the risk of metabolic related diseases, including type 2 diabetes, cardiovascular disease, fatty liver diseases and other human disease like female infertility, mental depression and even certain types of cancer. Obesity, characterized by excessive body weight, is often considered to result from an imbalance between energy intake and energy output. Either excessive food intake or insufficient physical activity can cause obesity. Abnormally elevated cholesterol, lipid and glucose levels due to disruption of metabolic hemostasis in obesity play causative roles in the development of metabolic diseases. Therefore, regulation of cholesterol, lipid and glucose levels in the body are critical for maintaining energy balance. During my Ph.D. thesis studies, I explored the function role of Farnesoid X receptor (FXR) and microRNA-34 (miR-34a), as key transcriptional and post-transcriptional regulators respectively, in maintaining metabolic hemostasis and energy balance. Nuclear receptors (NRs) are lipophilic ligand-dependent transcriptional factors that regulate the expression of specific target genes involved in diverse biological pathways, including development, differentiation, cell proliferation, reproduction and metabolism. FXR is a member of the nuclear receptor superfamily. After a meal, FXR is activated by bile acids (BA) and plays a crucial role in maintaining bile acid, cholesterol, glucose and lipid levels through gene regulation. In addition to these functions, previous genome-wide analysis of hepatic FXR binding sites by our laboratory revealed that binding of FXR was extensively detected in previously unknown categories of genes, including autophagy network genes and genes in the fibroblast growth factor-19 (FGF19) signaling pathway. MicroRNAs (miRs) are a class of small noncoding RNAs, usually 20-22 nucleotides long, first processed in the nucleus by the enzyme Drosha and then processed to its mature form in the cytoplasm by Dicer. MiRs usually function as negative post-transcriptional regulators by either inhibiting target protein translation or promoting degradation of target mRNAs by directly binding to the 3’UTR region of the mRNA. MiRs have been recognized as a distinct class of biological regulators with conserved functions and likely to target about 60% of mammalian genes. Moreover, aberrant expression of miRs occurs in many diseases, such as heart disease, cancer and obesity. In previous microRNA array analyses in wild-type (WT) mice and FXR knockout (FXR-KO) mice, our lab identified miR-34a as the hepatic miR most highly regulated by FXR and showed that FXR inhibits the expression of miR-34a indirectly through induction of the regulatory factor, Small Heterodimer Partner (Shp). Moreover, other researchers later showed that miR-34a is the most aberrantly elevated hepatic miR in obese mice and also is substantially elevated in diabetic human patients. Thus, in this thesis, my goal is to further understand the role of FXR and miR-34a as key metabolic regulators. There are four studies described in the following chapters: chapters 2 and 3 focus on aberrantly elevated miR-34a in liver and adipose tissue, respectively, in the obese state; chapters 4 and 5 are centered on the transcriptional regulation of FXR, repressing autophagy by inhibiting a fasted state regulator CREB and priming the liver for FGF19 signaling responses in the fed state, respectively.In chapter 2, I studied the function of hepatic miR-34a. As mentioned before, miR-34a levels are aberrantly elevated in obese mice and patients. My evidence indicated that βKlotho (βKL), a membrane co-receptor for FGF19 signaling, is a direct target of miR-34a. FGF19 is a fed-state hormone. After a meal, BA-activated FXR transcriptionally induces expression of FGF19 which is synthesized and secreted in intestine and acts at the liver where it binds to the membrane FGFR4/βKL receptor complex and mediates postprandial responses under normal physiological conditions. However,hepatic miR-34a is highly elevated in the obese state which results in repressed βKL expression, impaired FGF19-activated Erk signaling, and altered expression of FGF19 metabolic target genes. In vivo antisense inhibition of miR-34a in obese mice partially restored βKL levels and improved FGF19 target gene expression and resulted in beneficial metabolic outcomes such as weight loss and increased insulin sensitivity establishing the important role of miR-34a in metabolic disorders related to obesity.In chapter 3, I extended my studies on the function of miR-34a to adipose tissue, since βKL is also a co-receptor for another FGF, FGF21, which acts on adipose tissue to regulate metabolism. FGF21 is also an endocrine hormone that has lipid-lowering and insulin-sensitizing beneficial effects when administered to obese patients. FGF21 binds to the adipocyte membrane FGFR1/βKL receptor complex, and activates Erk signaling which results in induction of FGF21 metabolic target genes. Besides βKL, I identified FGFR1 as a direct target of miR-34a. Other researchers had already reported that expression levels of βKL and FGFR1 in adipose tissue are strikingly decreased in obesity. Obesity is a FGF21-resistance state, but the underlying mechanisms were not known. In this study, I found that aberrantly elevated adipocyte miR-34a in obesity downregulated both βKL and FGFR1 expression, thereby impairing FGF21 signaling. Moreover, miR-34a inhibited brown fat formation by suppressing the browning activators and attenuation of FGF21 signaling. This was an important observation since brown fat is known to increase energy expenditure by generating heat through uncoupled respiration so that promotion of brown fat would be desirable in obesity. Emerging evidence indicates that brown fat is substantially decreased while white fat is substantially increased in obesity, but the mechanisms underlying the decreased brown fat were unknown. Remarkably, I found that downregulation of miR-34a in obese mice dramatically increased beige depots (a form of brown fat produced in white fat that is distinct from classical brown fat), activated browning in brown adipose tissue, and reduced white fat. The substantial reduction in adiposity was associated with improved serum lipid profiles, increased mitochondria numbers and increased oxidative function. Collectively, the studies in chapter 2 and 3 revealed that miR-34a is a key factor that contributes to the FGF19-resistance and FGF21-resistance in liver and adipose tissue, respectively, that is observed in obesity. The striking beneficial results that I observed in vivo by inhibition of miR-34a provide evidence for the therapeutic potential of treating obesity with anti-miR-34a. In chapter 4, in collaboration with Dr. Sunmi Seok, a post-doctoral researcher in Kemper’s lab, I explored the functional role of hepatic FXR in autophagy, as a fed-state activated regulator. Autophagy is essential for cellular survival and homeostasis under nutrient-deprived fasting conditions, but must be suppressed in the nutrient-rich fed state. Short-term regulation of autophagy by nutrient-sensing kinases is well defined, but long-term transcriptional regulation is relatively unknown. Previous global ChIP-seq studies from our laboratory showed increased occupancy of FXR on genes in the autophagy network upon treatment with GW4064 (a synthetic FXR agonist). We showed that the expression level of those autophagy genes were decreased, which suggests that FXR may repress autophagy genes upon feeding. In an analysis of common sequences near the FXR repressed autophagy genes, the CREB binding motif was identified as a likely transcriptional factor that binds to their promoter regions. In a combined analysis of mouse hepatic FXR and CREB ChIP-seq data, 78 autophagy-related genes had shared FXR and CREB binding peaks among 230 genes in total, mostly in their promoter regions. Mechanistically, we showed that CREB upregulates autophagy genes by recruiting its coactivator CRTC2 under fasting conditions. Upon feeding, bile-acid activated FXR trans-repressed these autophagy genes by disrupting the functional CREB/CRTC2 complex.This study identifies the FXR/CREB axis as a novel, key physiological switch regulating autophagy that results in sustained nutrient regulation of autophagy during feeding/fasting cycles.Hepatic metabolic responses to a meal are regulated by an intricate interplay among intestinal, pancreatic and hepatic hormones and factors. In chapter 5, I examined whether FXR, which induces FGF19 synthesis in the intestine, also primes the liver to enhance FGF19 signaling in the fed state. Our previous hepatic FXR Chip-seq data revealed that FXR has binding sites on FGF19 signaling component genes, especially the membrane co-receptor βKL. My preliminary data showed that FXR occupancy and expression of βKL increased after activation of FXR by GW4064 treatment. In addition, I showed that FXR-RXRα bind to the βKL gene by in vitro gel shift assays and that FXR up-regulates βKL by reporter-luciferase assays. More interestingly, my initial in vivo time course studies showed that FXR regulates FGF19 synthesis, mostly from intestine, followed by the induction of βKL gene expression in the liver. Future studies will be needed to definitively test the hypothesis that FXR primes the liver for FGF19 function.In summary, in this thesis, I studied the functions of miR-34a and FXR as key metabolic regulators in modulating FGF19 and FGF21 signaling, stimulating browning of fat, inhibiting autophagy, and priming the liver for response to signals from the gut. I hope that the findings from the miR-34a and FXR projects in my thesis reveal new potential targets for both diagnosis and treatment of metabolic diseases.
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Metabolic regulation by nuclear receptor FXR and micro RNA-34a in health and disease
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