Date of Award

2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmaceutical Sciences

First Advisor

Angela Slitt

Abstract

Obesity and diabetes are the most prevalent metabolic disorders. It is estimated that more than 86% of US adults would be overweight or obese and more than 50% obese by the year 2030. Non Alcoholic Fatty Liver Disease (NAFLD) has also been referred to as hepatic manifestation of insulin resistance. NAFLD or steatosis is defined as lipid accumulation exceeding 5% by weight in hepatocytes in the absence of substantial alcohol intake. The prevalence of NAFLD is estimated to be between 17% and 40% of the world population, known to afflict obese and normal weight persons and children as well. Epidemiological and clinical studies demonstrate changed pharmacokinetic and –dynamic parameters of drugs and environmental toxicants in models of NAFLD and cause drug induced liver injury (DILI) which can be attributed to altered expression of Phase I, Phase II drug metabolizing enzymes and drug transporter expression in human and rodent models of NAFLD. NAFLD also causes propogation of insulin resistance complicating metabolic syndrome. Due to its increasing prevalence throughout the world, understanding mechanisms and consequences of NAFLD and identifying novel targets to reverse this condition are of increasing clinical relevance.

Nuclear factor-E2 related factor 2 (Nrf2) belongs to leucine zipper based loop-helix-loop family of transcription factors which plays an important role in activation of a battery of antioxidant genes, thus protecting the cells from oxidative stress which is either causality or a consequence that aggravates a number of disorders, an important condition being NAFLD. Nrf2 is sequestered in the cytoplasm via sulfhydryl based interactions with Kelch like ECH-associated protein 1 (Keap1); oxidative stress disrupts these interactions and phosphorylation based changes in Nrf2 protein aid its translocation into the nucleus where it binds to antioxidant response element (ARE) and initiates transcription of the antioxidant gene battery which includes but not limited to NAD(P)H:quinine oxidoreductase 1 (NQO1), hemoxygenase (HO1), glutamine cysteine ligase catalytic and regulatory subunit (GCLC and GCLM). Hepatic clearance is a predominant mechanism for excretion of chemicals, such as hormones and drugs. Several families of transporters localized to sinusoidal and canalicular membrane of hepatocytes drive bile flow and biliary excretion. Nrf2 also regulates expression of ATP-binding cassette (ABC) superfamily of membrane proteins, efflux organic anions into bile (e.g. Abcc2) and from hepatocytes into blood (e.g. Abcc3 and 4), which contribute to hepatic clearance of various endogenous and exogenous chemicals.

Diet (caloric restriction, CR) and exercise are the recommended therapeutic intervention to treat NAFLD and reverse hepatic fat accumulation. Beneficial effects of CR are attributed to activation of Sirtuin 1, a NAD (+)-dependent protein deacetylase which activates gluconeogenic and fatty acid oxidation genes expression via coactivator; Peroxisome proliferator-activated receptor gamma coactivator 1-alpha. The important regulatory roles of SIRT1 in both lipid and glucose metabolism make it an important molecule in hepatic mechanisms in development of steatosis.

Resveratrol, an antioxidant compound and a potent Sirt1 activator also activates Nrf2 in certain in vitro models as determined by an increase in the expression of prototypical Nrf2 target genes. Additionally, Nrf2 has been implicated in protection against development of cancer in mice undergoing CR. Ability of CR mimetics or Sirt1 activators to activate Nrf2-Keap1 pathway and scant but significant evidence of Nrf2 in nutrient regulation demand an indepth analysis of the Nrf2-Keap1 pathway in the nutrient deprived conditions.

A possible role of Nrf2 in adipogenesis, adipocyte differentiation as well as glucose tolerance has been suggested in mouse models, however, very little is known about Nrf2 signaling pathway in nutrient deprived states. Very little is known about effect of nutritional status on biliary clearance mechanisms or bile flow.

The purpose of study performed in MANUSCRIPT I is to elucidate the effect of fasting pathways on Nrf2-Keap1/ARE pathway. Few studies exist regarding how fasting affects transporter or the nuclear receptor-regulated pathways which regulation expression. Fasting effects on the Nrf2-Keap1 signaling pathway, which is responsive to oxidative stress and altered redox status have not been elucidated. The observations presented in this study prove that 1) fasting increases Abcc expression through Nrf2- and Sirt1-dependent mechanisms, 2) cAMP/PKA activators increase ARE activation via Nrf2- and Sirt1-dependent mechanisms in vitro, 3) Nrf2 expression is co-ordinately regulated with Pgc-1α and Sirt1 expression and 4) obesity/steatosis diminishes fasting-mediated Abcc induction.

In summary, MANUSCRIPT-I demonstrates that fasting and PKA activators increase mouse Abcc2-4 expression in liver by Nrf2- and Sirt1-related mechanisms in mouse liver and hepatocytes. Additionally, 8-Br-cAMP and PKA activating compounds increase Nrf2 target gene and ABCC2-4 mRNA expression in human hepatocytes. Our data illustrate an undescribed role for Nrf2 as a “fasting” responsive transcription factor that is activated via cAMP/PKA-Sirt1 upstream mechanisms.

CR alters activity and expression of various biotransformation enzymes such as Sult2a1, Cyp2b10, Ugt1a1, CYp4a14 in liver. However, to our knowledge, no studies have shown how CR actually affects drug transporter expression in livers of obese mice, which have hepatic steatosis and better mimic the population most likely to undergo intervention. The purpose of study performed in MANUSCRIPT II hypothesized that CR would reverse the NR and drug transporter expression changes previously observed in obesity-induced hepatic steatosis. Given the well-defined association of Nrf2 with Abcc transporter induction, emphasis was placed on the Nrf2 pathway, but other NRs that have been described to regulate transporter expression were also evaluated. Our data herein illustrate that CR differentially regulates NR, biotransformation enzyme, and transporter expression in livers from C57Bl/6 (WT) and Lepob/ob (OB) mice. Furthermore, AMPK and Sirt1 activators differentially modulated transporter and NR expression in lean and steatotic hepatocytes.

To summarize, MANUSCRIPT II demonstrates that activation of CR pathways can only partly reverse the changes in drug metabolizing enzyme and transporter expression occurring due to fat deposition in the liver. Leptin appears to play an important role in upstream regulation of pathways that relay CR signals to ultimately change DME and DT expression. The above observations also indicate that the inability of CR to reverse certain DME and DT changes may indicate towards altered disposition and pharmacokinetics of metabolites and xenobiotics. Data also shows that CR decreases the expression and activity of Nrf2-ARE regulatory pathway and target gene expression.

Small non-coding RNAs, microRNAs have been recently demonstrated to provide an additional layer of regulatory control in development, homeostasis and pathology. miRNA expression and regulation are being explored in detail as potential targets and possible biomarkers in various diseases, specifically metabolic syndrome and cancer. Various miRNAs such as miR34a, miR122, miR370, miR221, miR33a, let-7 have been proven to be important regulators of fatty acid oxidation and lipid synthesis regulators such as Sirt1, Pgc-1α, Srebp1c, Lxr. In MANUSCRIPT III we demonstrate differential susceptibility of a mouse model, constitutively overexpressing Nrf2 (Keap1-KD mice) to CR. We attribute these changes to novel changes in the miRNA regulatory circuit.

We demonstrate that the enhanced susceptibility to CR in Nrf2 overexpressing mice could be partly associated with differential changes in miRNA expression such as miR34a, miR370, miR122 and miR144, especially those known to be involved in regulation of lipogenic as well as fatty acid oxidation genes.

In summary, the studies presented herein demonstrate Nrf2-ARE pathway is a nutrient sensitive pathway that is: (a) activated upon fasting via Sirt1-Pgc-1α dependent mechanisms, (b) negatively regulated upon CR and AMPK pathway, (c) plays a role in development of obesity, and (d) regulates expression of miRNA regulatory circuit thereby changing susceptibility to weight loss and fatty acid metabolism, (e) is a potential target to alleviate metabolic syndrome.

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