Rnase L Mediates Glucose Homeostasis Through Regulating The

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RNASE L MEDIATES GLUCOSE HOMEOSTASIS THROUGH REGULATING THE

INSULIN SIGNALING PATHWAY

Danting Liu

Bachelor of Chemical Engineering

Nanjing University of Technology

June 2008

Submitted in partial fulfillment of requirements for the degree

DOCTOR OF PHILOSOPHY IN CLINICAL AND BIOANALYTICAL CHEMISTRY

at the

CLEVELAND STATE UNIVERSITY

December 2018

We hereby approve this dissertation for

Danting Liu

Candidate for the Doctor of Philosophy in Clinical-Bioanalytical Chemistry degree for

the Department of Chemistry

and the CLEVELAND STATE UNIVERSITY’S

College of Graduate Studies by

Committee Chairperson, Dr. Aimin Zhou Department of Chemistry

Date

Committee Member, Dr. Michael Kalafatis Department of Chemistry

Date

Committee Member, Dr. Bin Su Department of Chemistry

Date

Committee Member, Dr. Xue-Long Sun Department of Chemistry

Date

Committee Member, Dr. Nolan Holland Department of Chemistry

Date

Date of Defense: October 17th, 2018

ACKNOWLEDGMENT

First and foremost, I would like to thank my supervisor, Dr. Aimin Zhou, for his guidance, encouragement, and constructive criticisms throughout my doctoral studies. His passion for research is infectious and inspires me to be a better scientist every day. This thesis would not have been possible without his guidance and support. Dr. Zhou is a great mentor and I am honored to have worked under his supervision.

I would also like to give my greatest gratitude to my committee members: Dr.

Michael Kalafatis, for the critical insights he has provided in both program direction and research advising; Dr. Bin Su, for his continuous support of my work and the generosity to use his facilities to help me successfully finish my project; Dr. Xue-long Sun, for his help, support, advice, encouragement and patience; Dr. Nolan Holland, for his valuable comments and suggestion on my research progression.

In addition, I would like to thank Dr. Xiang Zhou, who trained me in the field of mass spectrometric analysis and offered developmental coaching to elevate my career.

Moreover, I am pleased to have the opportunity of close collaboration with Dr. Yanqing

Zhang and her group in Tianjin University of Commerce. Dr. Zhang’s conscientiousness and enthusiasm in research are admirable affecting me to develop my image.

Furthermore, I would also like to thank those friends and colleagues that have shared in my scientific journey, especially the past and current lab members in Dr. Zhou’s lab. I enjoy the friendly working environment and appreciate their technical help, group discussion, and friendship.

Last but not least, I am forever grateful to my family. To my late grandparents, I missed so much of your lives and I wish you were still here. I am indebted to you all. To

my parents, I am so fortunate to receive your unconditional love and support, and this thesis is dedicated to you. I would also like to thank my cousin, Yang Zhang who helped and supported me a lot during my doctoral training.

Thank you to all of you who have made this possible.

RNASE L MEDIATES GLUCOSE HOMEOSTASIS THROUGH REGULATING THE

INSULIN SIGNALING PATHWAY

DANTING LIU

ABSTRACT

Diabetes is characterized by hyperglycemia mainly due to defect in insulin secretion and/or action. Regulation of glucose transport and use by insulin is central to the maintenance of whole-body glucose homeostasis. One of the potential mechanisms associated with insulin sensitivity is the activation of insulin receptor (IR) and subsequently transduces the signal through phosphorylation of insulin receptor substrate (IRS) and activation of the PI-3K/Akt pathway. In contrast, activation of the mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (p70S6K) suppresses the signal cascade. RNase L, an interferon (IFN)-inducible enzyme, plays an important role in IFN functions against viral infection and cell proliferation. However, a direct link between

RNase L and insulin sensitivity has yet to be clearly established. RNase L+/+ and -/- mouse embryonic fibroblasts (MEFs) and hepatocytes were used to investigate the role of RNase

L in insulin signaling and sensitivity. Cells were treated with insulin at various time points and different concentrations. Activation of the insulin signaling pathway was determined by immunoblot analyses for the protein level and phosphorylation status of these components such as IR/p-IR, IRS1/p-IRS1 and AKT/p-AKT in the presence or absence of a chemical inhibitor. Interestingly, we found that RNase L might mediate insulin signaling and glucose homeostasis through impacting insulin receptor (IR) which is a transmembrane receptor activated by insulin. The phosphorylation status of IR was significantly reduced in the cell deficient RNase L. As a result, activation of downstream components

vi in the insulin signaling pathway and the PI3K/AKT pathway was significantly inhibited in

RNase L-/- cells. Further investigation of the molecular mechanism underlying the role of

RNase L in mediating the activation of IR revealed that RNase L might regulate cleaving the precursor of IR and activating IRS-1 via the ubiquitin/ proteasome system. Our results obtained from this study provide a better understanding of RNase L functions and open a new horizon for its role in the metabolic system besides in the defense of viral infection.

vii TABLE OF CONTENTS

Page

ACKNOWLEDGMENT...... iv

ABSTRACT ...... vi

LIST OF TABLES ...... xiii

LIST OF FIGURES ...... xiv

ABBREVIATIONS ...... xvi

CHAPTER ...... 1

I. INTRODUCTION OF PROJECT I

1.1 Ribonuclease L...... 1

1.1.1 2'-5' oligoadenylate (2-5A) / RNase L system ...... 2

1.1.2 The structural properties of RNase L ...... 6

1.1.3 The biological functions of RNase L ...... 10

1.1.3.1 Antiviral activities of RNase L ...... 10

1.1.3.2 RNase L mediates cell apoptosis ...... 12

1.1.3.3 RNase L regulates cellular proliferation ...... 13

1.1.3.4 RNase L is a tumor suppresor ...... 14

1.1.3.5 The involvement of RNase L in the immune

system ...... 15

1.1.3.6 RNase L effects lipid metabolic ...... 18

1.2 Overview of Diabetes Mellitus (DM) ...... 18

1.2.1 Type I Diabetes Mellitus ...... 19

1.2.2 Type II Diabetes Mellitus ...... 20

viii 1.2.3 Obesity and Type II Diabetes ...... 20

1.2.3.1 Overview of Obesity ...... 20

1.2.3.2 Linking of Obesity to Diabetes ...... 21

1.3 Insulin signaling pathway ...... 21

1.3.1 Insulin Receptor ...... 24

1.3.2 Insulin receptor substrate ...... 26

1.3.3 Phosphatidylinositol-3 kinase ...... 28

1.3.4 PKB/AKT pathway ...... 29

1.3.5 Glucose transporter ...... 33

1.4 The Physiological Effects of Insulin ...... 34

1.4.1 Liver ...... 34

1.4.2 Skeletal Muscle ...... 34

1.4.3 Adipose Tissue ...... 35

1.5 Insulin Resistance ...... 35

1.6 Rationale for the Studies ...... 36

1.7 Specific Aims and Hypotheses ...... 38

II. THE ROLE OF RNASE L IN INSULIN SIGNALING PATHWAY ...... 39

2.1 Introduction ...... 39

2.2 Material and method ...... 44

2.2.1 Experimental animals...... 44

2.2.2 Experimental cell lines ...... 45

2.2.2.1 Primary Mouse embryo fibroblasts (MEFs) ...... 45

2.2.2.2 BC 10 T-ag cell line ...... 45

ix 2.2.2.3 SK-Hep-1cell line ...... 46

2.2.3 Isolation and of primary Mouse embryo fibroblasts ...... 46

2.2.4 Isolation of primary mouse hepatocytes ...... 46

2.2.5 RNase L knockout in SK-Hep-1 cell line ...... 47

2.2.5.1 Introduction of the CRISPR/Cas 9 method ...... 47

2.2.5.2 RNase L gene knockout ...... 49

2.2.6 Cell culture and insulin treatment ...... 49

2.2.7 Sample preparation ...... 50

2.2.8 Western blot analysis ...... 50

2.3 Results ...... 52

2.3.1 Deficiency of RNase L attenuates the insulin signaling

pathway in MEFs ...... 52

2.3.2 RNase L affects the expression and activation of Insulin

Receptor in MEFs ...... 55

2.3.3 Effects of RNase L on Insulin Signaling in Primary

Hepatocytes ...... 58

2.3.4 RNase L mediate insulin signaling in SK-HEP-1 cells ..60

2.4 Discussion ...... 63

III. INVESTIGATE THE MECHANISM BY WHICH RNASE L REGULATES

THE INSULIN SIGNALING PATHWAY ...... 65

3.1 Introduction ...... 65

3.1.1 P97/VCP system ...... 66

3.1.2 RNase L impacts phosphorylation of P97/VCP ...... 67

x 3.1.3 The ubiquitin/proteasome pathway and its impact of

IRS-1 ...... 68

3.2 Material and method ...... 68

3.2.1 Experimental cell line and cell culture ...... 68

3.2.1.1 L929 PD12 cell line ...... 68

3.2.1.2 Cell culture and insulin treatment ...... 71

3.2.2 MG132 treatment on MEFs ...... 71

3.2.3 Western blot analysis ...... 74

3.3 Results ...... 75

3.3.1 Overexpression of the 2-5A binding domain affects the

insulin signaling pathway ...... 75

3.3.2 RNase L impacts phosphorylation of P97/VCP and

ubiquitination in MEFs ...... 79

3.3.3 Inhibiting the proteasome enhanced the activation of

IRS-1 in RNase L deficient MEFs ...... 81

3.4 Discussion ...... 83

IV. SUMMARY AND FUTURE DIRECTION ...... 86

4.1 Summary of the project ...... 86

4.2 Future direction ...... 87

4.2.1 Determine the role of RNase L in insulin sensitivity in

the adipose tissue and cells ...... 87

4.2.2 Investigate the effects of RNase L in insulin signaling

in vivo ...... 88

xi 4.2.3 Test the kinase function of RNase L ...... 88

4.2.4 Examine the RNase L level in the samples of metabolic

disorders patients ...... 89

BIBLIOGRAPHY ...... 90

xii LIST OF TABLES

Table Page

I. Table 2.1 Antibodies I...... 51

II. Table 3.1 Antibodies II ...... 74

xiii LIST OF FIGURES

Figure Page

1. 1.1 2-5A/RNase L system ...... 3

2. 1.2 Structure of the 2-5A: 2’-5’ oligoadenylates tetramer ...... 4

3. 1.3 Functional model for the activation of RNase L by 2-5A ...... 5

4. 1.4 The structure of RNase L ...... 8

5. 1.5 Crystal structure of an ankyrin repeat domain complexed with

2-5A ...... 9

6. 1.6 The 2–5A/RNase L pathway ...... 17

7. 1.7 Insulin-signaling pathways ...... 23

8. 1.8 Insulin Receptor ...... 25

9. 1.9 Binding site of IRS proteins ...... 27

10. 1.10 Molecular Mechanisms of AKT Regulation ...... 30

11. 1.11 Schema of the PI3K/AKT pathway ...... 32

12. 1.12 Effect of RNase L on diet-induced obesity ...... 37

13. 2.1 Involvement of RNase L in lipid metabolism ...... 41

14. 2.2 RNase L mediates activation of ERK and AKT ...... 42

15. 2.3 Effect of RNase L on phosphorylation of p70s6k ...... 43

16. 2.4 The principle of CRISPR/Cas9-mediated gene disruption ...... 48

17. 2.5 Effects of RNase L on activation of IRS-1 and Akt in MEFs ...... 53

18. 2.6 Effects of RNase L on activation of IRS-1 and Akt in primary MEFs ...... 54

19. 2.7 Effect of RNase L on the activation of IR in MEFs ...... 56

20. 2.8 Effect of RNase L on the activation of IR in primary MEFs ...... 57

xiv 21. 2.9 Effect of RNase L on Insulin Signaling in Primary Hepatocytes ...... 59

22. 2.10 Knockout of RNase L in liver cancer cells ...... 61

23. 2.11 RNase L mediates insulin signaling in SK-HEP-1 cells ...... 62

24. 3.1 Expression of dominant negative RNase L in L929 cells ...... 70

25. 3.2 The inhibitory role of MG132...... 73

26. 3.3 Overexpression of the 2-5A binding domain affects the expression

of IR ...... 76

27. 3.4 Overexpression of the 2-5A binding domain affects the insulin signaling ...77

28. 3.5 RNase L medicates phosphorylation of P97/VCP and ubiquitination

in MEFs ...... 80

29. 3.6 Inhibition of the proteasome system enhanced the activation

of IRS-1 ...... 82

xv ABBREVIATIONS

2-5A 2'-5' oligoadenylate 4E-BP1 4Ebinding protein 1 ARD ankyrin repeat domains BMI Body mass index CFS chronic fatigue syndrome DM Diabetes Mellitus dsRNA double-stranded RNA EMCV Encephalomyocarditis virus ER endoplasmic reticulum ERAD Endoplasmic-reticulum-associated protein degradation FBS fetal bovine serum GAB1 Grb-2-associated protein GM-CSF granulate macrophage colony stimulating factor GS glycogen synthase GSK Glycogen synthase kinase HPC hereditary prostate cancer HSV herpes simplex virus IFN Interferon IR Insulin Receptor IRS Insulin receptor substrate JNK Jun N-terminal kinases KEN kinase-extenstionnuclease MEF mouse embryonic fibroblasts MetS metabolic syndrome MFS major facilitator superfamily mTOR mammalian target of rapamycin NAFLD nonalcoholic fatty liver disease OAS 2'-5' oligoadenylate synthetases p70S6K ribosomal protein S6 kinase

xvi PDK-1 Phosphoinositide-dependent kinase-1 PEPCK phosphoenolpyruvate carboxykinase PI3K phosphoinositide 3-kinase PK Protein kinase PKB protein kinase B PVDF polyvinylidene difluoride RNase L Ribonuclease L RTK receptor tyrosine kinase SDS sodium dodecyl sulfate SH2 SRC homology 2 SV40 simian virus 40 T1DM Type I Diabetes Mellitus T2DM Type II Diabetes Mellitus TSC tuberous sclerosis complex protein UPR unfolded protein response VCP Valosin-containing protein

xvii

CHAPTER I

INTRODUCTION

1.1 Ribonuclease L

Ribonuclease L (RNase L), an interferon (IFN)-inducible endoribonuclease, is one of the key enzymes in the 2-5A system of IFN action against viral infection and in the control of cellular proliferation [1, 2]. RNase L is localized in both nuclei and cytoplasm, and widely expressed in mammalian cells from mouse to humans [3, 4]. The expression of

RNase L is found in all organs, but significantly higher in the intestine, spleen, thymus, lung and testis. The protein level of RNase L reaches its peak at 5 days of age and then gradually decreases, except in the organs mentioned above where it remains highly expressed in adulthood [5]. Actually, the cell growing status also affects the expression level of RNase L, for example, up-regulation of RNase L was observed in the cells under differentiation and growth arrest [6]. The successfully cloning of RNase L in 1993 allows the detailed elucidation of its remarkable structural, functional and physiological characteristics in diseases [7].

1 1.1.1 The 2'-5' oligoadenylate (2-5A) system

The 2-5A system is an RNA degradation pathway induced by IFNs. The 2-5A pathway consists of three important components, 2-5A synthetases (OAS), 2-5A degrading enzyme, and RNase L (Figure 1.1) [8]. IFN induces the transcription of several 2-5A synthetase genes. OAS can be activated by binding a double-stranded RNA (dsRNA), frequently generated from virus infection. After activation, 2-5A synthetases converts ATP to PPi and a series of short 2', 5'- linked oligoadenylates referred to as 2-5A molecules

(Figure 1.2) with the formula [ppp5'A(2'p5'A )n; n ≥ 2] [9]. 2-5A binds RNase L with high affinity, converting it from its inactive, monomeric state to a potent dimeric endoribonuclease (Figure 1.3). Activated RNase L degrades single-stranded viral and cellular RNAs, as a result, viral replication and cell proliferation are inhibited [10]. It is believed that 2-5A activated RNase L cleaves RNAs within single-stranded regions, preferentially after UpUp and UpAp dinucleotides [12]. The 2-5A system mainly mediates host defense against certain types of viral infection. However, studies have also revealed that the 2-5A system is involved in a wide board of other biological functions, such as in cell growth and differentiation, heat shock, atherosclerotic plaque, pathogenesis of type I diabetes, and apoptosis were also reported [13]. 2-5A accumulates and RNase L is activated in intact infected cells. Cells overexpressing RNase L overcome viral infection [14]. In contrast, overexpression of a dominant negative mutant of RNase L results in increased susceptibility to viral infection [15]. In vivo studies also show that mice containing targeted disruption of RNase L gene succumb to encephalomyocarditis (EMCV) infection more rapidly than infected wild type mice [8].

Figure 1.1 2-5A/RNase L system

(Chakrabarti A, Jha BK, Silverman RH. J Interferon Cytokine Res. 2011)

Figure 1.2 Structure of the 2-5A: 2’-5’ oligoadenylates tetramer

(Bisbal C, Silverman RH. Biochimie. 2007)

Figure 1.3 Functional model for the activation of RNase L by 2-5A

(Silverman RH. Biochemistry. 2003)

5 1.1.2 The structural properties of RNase L

The cDNA of human RNase L gene encodes an 84 kDa protein with 741 amino acids [1]. The structural analysis of RNase L has revealed that RNase L is composed of two parts (Figure 1.4): the N-terminal half contains ankyrin repeat domains (ARD) which function as a repressor. The C-terminal half has a kinase-extension-nuclease (KEN) domain consisting of a protein kinase (PK)-like domain and a ribonuclease domain

(RNASE) with ribonuclease activity [16].

There are 9 ankyrin repeats, among them, eight are complete (R1-R8) and one is a partial repeat (R9) that appears as a disordered segment in the crystal structure of the ARD of human RNase L [17]. The unique feature of the ARD in RNase L demonstrates that repeats 2 to 4 is the binding site of 2-5A [17]. During the activation of RNase L, the N terminal domain functions as a repressor of the ribonuclease domain in the C terminal domain (Figure 1.3). Actually, three ankyrin repeats, 7, 8, and 9 [18, 19] are responsible for the minimum repression. So far RNase L is the only nuclease known to contain ARD, the typical protein-protein interaction domains, suggesting that RNase L may interact with other proteins [9,16].

The C-terminal half of RNase L has a cysteine rich region with a protein kinase homology, suggesting that RNase L may have specific kinase activity, although it has not been demonstrated to date. The protein kinase-like and ribonuclease domains of RNase L are homologous with Ire1, which is also a kinase and an endoribonuclease that functions in the unfolded protein response (UPR) from yeast to humans [16, 19, 20]. The function of

KEN domain in RNase L is associated with dimerization and catalysis. RNA can be cleaved in the absence of 2-5A by an isolated C-terminal domain of RNase L [18, 21]. In that case,

6 the RNASE domain becomes constitutively active after removal of the ARD (although at

6-fold reduced activity compared with activated full-length protein) [18]. Apparently, the

ARD is important in regulating the activity of RNase L. The binding of 2-5A to the N- terminal half probably induces a conformational change of the enzyme, causing the N- terminal repressor domain to release from the C terminal ribonuclease domain, and unmasks the dimerization domain (Figure 1.3).

Figure 1.4 The structure of RNase L

(Chakrabarti A, Jha BK, Silverman RH. J Interferon Cytokine Res. 2011)

Figure 1.5 Crystal structure of an ankyrin repeat domain complexed with 2-5A

(Tanaka N,Nakamura KT. EMBO J. 2004)

(A) Structural and functional domains of RNase L. Ankyrin repeats are shown starting with blue at repeat 1 and ending with red at repeat 8. (B) Structure of the predominant trimeric species of 2-5A. (C, D) Surface (top) and ribbon (bottom) representations of the ARD/2-5A complex. Ankyrin repeats (R1–R8) are shown as in (A). The bound 2-5A molecule is shown as a ball-and-stick model. The view in (D) was obtained by rotating the view in (C) by 90°.

9 1.1.3 The biological functions of RNase L

The biologic activities of RNase L have been extensively investigated [22]. As an intracellular RNA regulator, RNase L is involved directly and indirectly in a wide range of physiologic activities. Disruption of RNase may lead to an altered physiological state associated with diseases. The role of RNase L in antiviral activities, apoptosis, cellular proliferation, and the potentially involved mechanisms is discussed below.

1.1.3.1 Antiviral activities of RNase L

The classical characteristic of RNase-L is to function as an antiviral effector of

IFNs action against viruses [23]. The most compelling evidence is the accumulation of 2-

5A and activated RNase L in virus-infected cells [24]. The replication of encephalomyocarditis virus (EMCV), a member of picornaviruses causing mild febrile illness in a number of species including humans, can be effectively suppressed by the activation of the 2-5A system. In EMCV- and IFN- treated cells, accumulation of 2-5A and rRNA cleavage were observed, suggesting that both OAS and RNase L have been activated, resulting in RNA degradation [25]. Increased levels of resistance to EMCV infection occurred in human glioblastoma T98G cells stably transfected with a human OAS cDNA

[26]. In other studies, transfection of murine cDNAs encoding the 43-kDa OAS into NIH

3T3 cells resulted in varying levels of constitutive OAS production and greatly increased resistance to EMCV infection [27]. It has been reported that the antiviral effect of IFN-α/β for EMCV was reduced by 250-fold when expression of the SVT2/ZB1 mutant, that lacks

RNase enzymatic activity, prevented 2-5A-dependent rRNA cleavage [15]. After truncation of RNase L by 89 amino acids at the C-terminal residues, its catalytic activity

10 was completely abolished as demonstrated in stably expressed murine SVT2 cells and inhibited the 2-5A system. Studies have also found an increase of 2-5A levels, activation of RNase L and characteristic rRNA breakdown in vaccine-virus infected and IFN-treated cells [28].

The induction of an antiviral state by type I IFNs was evaluated in primary trigeminal ganglion cells using herpes simplex virus type 1 (HSV-1). The cells treated with mouse IFN-β showed the greatest resistance to HSV-1 infection. In the cells treated with

IFN-α and IFN-β, suppression of HSV-1 replication occurred through an RNase L dependent pathway [29]. Over expressed RNase L by >100-fold compared with levels of the endogenous murine RNase L in 3T3/pLZ suppressed the replication of different viruses including EMCV, vesicular stomatitis virus, human parainfluenza virus-3, and vaccinia virus. Pre-treatment of the cells with IFN remarkably increased their capability against viral infection. The 2-5A system is also activated in the cells with HIV infection [30]. The

HIV-1 gene product Tat drives a dramatic increase in the steady-state levels of HIV-1 long terminal repeat (LTR)-derived mRNAs. The interaction of tat with a region in the HIV

LTR (TAR) mediates up-regulation of HIV mRNAs. Interestingly, the TAR RNA sequence activates OAS [31], resulting in an increase of 2-5A oligonucleotides, subsequently activates RNase L and inhibits the replication of HIV [32]. It has been postulated that HIV- trans–activated OAS suppressing HIV replication could lead to design a possible therapeutic approach for AIDS treatment.

11 1.1.3.2 RNase L mediates cell apoptosis

A line of evidence has demonstrated that RNase L is involved in cell apoptosis in an IFN-dependent and independent fashion. An infected cell undergoes apoptosis is a natural defense way to prevent viral progeny from dissemination. Obviously, the activation of the 2-5A system may contribute to the event. For example, activation of RNase L in 2-

5A transfected cells results in specific 18S rRNA cleavage and induction of apoptosis, as measured by TUNEL and annexin V binding assays [6]. Overexpression of RNase L by using a recombinant vaccinia viral vector induces apoptosis in mammalian cells and coexpression of a 2-5A synthetase in the same cells promotes the event [34]. It has been demonstrated that the Jun N-terminal kinases (JNK)-dependent stress response is responsible for efficient induction of apoptosis initiated by RNase L through a release of cytochrome c from mitochondria and subsequently activation of the caspase cascade [35].

DU145 cells, a prostate cancer cell line, were resistant to apoptosis when the expression of

RNase L was down-regulated by siRNA [36]. In animal models, RNase L null mice showed enlarged thymuses caused by a reduced level of spontaneous apoptosis in thymocytes.

Furthermore, thymocytes and MEF isolated from RNase L null mice are resistant to apoptosis induced by staurosporine and irradiation [37]. Interestingly, a number of evidence suggest that RNase L could participate in an apoptotic pathway in the absence of

2-5A, implicating the existence of an alternative pathway independent in the IFN-induced

2-5A/RNase L system. However, how RNase L mediates the IFN-independent apoptosis remains to be fully understood.

12 1.1.3.3 RNase L regulates cellular proliferation

RNase L also has an impact on the cell proliferation. Analysis of the levels of the components in the 2-5A/RNase L system reveals an inverse correlation between 2-

5A/RNase L pathway activities and cell proliferation. For example, RNase L and OAS activities were elevated in arrested or differentiated cells as well as in the cells treated with an antiproliferative agent, indicating RNase L is involved in the fundamental control of cell proliferation and differentiation [38]. Ectopic expression of OAS or RNase L resulted in cell quiescence, senescence or apoptosis, and the induction of apoptosis and senescence was reduced when RNase L is absent [39]. Cells expressing a dominant negative RNase L were resistant to the antiproliferative activity of IFN-α. In contrast, it has been reported that the over expression of RNase L in murine NIH 3T3 cell increased IFN-α antiproliferative function. Furthermore, introduction of 2-5A into the cells also results in an inhibition of growth rates, suggesting that RNase L may regulate cell growth [40].

Similar to the role of RNase L in cell apoptosis, RNase L can regulate cell proliferation independent of IFN-α. Without IFN-α treatment, RNase L-/- MEF cells grew 1.6-fold faster compared with RNase L+/+ MEF cells. The growth rate of bone marrow cells isolated from

RNase L-/- mice was 1.73-fold faster than that from RNase L+/+, when granulate macrophage colony stimulating factor (GM-CSF) was present, suggesting that RNase L regulating cell proliferation may be through other pathways as well. RNase L was also found to inhibit fibrosarcoma growth in nude mice [41].

13 1.1.3.4 RNase L is a tumor suppressor

Over-expression of RNase L in NIH 3T3 cells markedly enhances the antiproliferative function of IFN, whereas the dominant-negative RNase L suppresses the anti-proliferative activity of IFN in SVT2 cells [15]. The 2-5 A/RNase L system contributes to apoptosis of viral infected cells by both degrading cellular RNA and blocking anti- apoptotic proteins such as Bcl-2 [28]. Proapoptotic function of RNase L may prevent some diseases such as prostate cancer. Several studies have linked the hereditary prostate cancer

1 (HPC1) allele to the RNase L gene located at chromosome 1q25, a region deleted or rearranged in some breast cancers [42,43,44]. The studies suggest that mutations in RNase

L predispose men to an increased incidence of prostate cancer. The RNase activities of missense variants of human RNase L were compared after expression in a mouse RNase L

-/- cell line. Several variants (G59S, I97L, I220V, 322F, Y529C, and D541E) produced similar levels of RNase L activity as a wild-type enzyme, but the R462Q variant, previously implicated in up to 13% of unselected prostate cancer cases, bound 2-5A at wild-type levels but had a 3-folds decrease in RNase L activity. The deficiency in RNase L (R462Q) activity was correlated with a reduction in its ability to be dimerized into a catalytically active form.

Furthermore, the RNase L mutant (R462Q) was defective in causing apoptosis in response to 2-5A consistent with its possible role in prostate cancer development [45]. It has been proposed that RNase L functions as a tumor suppressor on the basis of its ability to degrade

RNA, thus initiating a cellular stress response that leads to apoptosis. The anti-tumor activity of RNase L can be induced by a small molecule, 2-5A. Indeed, 2-5A activation of

RNase L produces a remarkable stimulation of transcription (>/=20-fold) for genes that suppress virus replication and prostate cancer [46]. Among the 2-5A-induced genes are

14 several IFN-stimulated genes, including IFN-inducible transcript 1/P56, IFN-inducible transcript 2/P54, IL-8, and IFN-stimulated gene 15. 2-5A also potently elevated the expression of macrophage inhibitory cytokine-1/non-steroidal anti-inflammatory drug- activated gene-1, a TGF-beta super family member implicated as an apoptotic suppressor of prostate cancer. Transcriptional signaling to the macrophage inhibitory cytokine-1/non- steroidal anti-inflammatory drug-activated gene-1 promoter was deficient in HeLa cells expressing a nuclease-dead mutant of RNase L, which was JNK and ERK dependent, and both were activated in response to 2-5A treatment. These findings suggest that activation of RNase L by 2-5A may produce antitumor response as demonstrated in a mouse model of prostate cancer [45]. 2-5A itself also induces apoptosis in prostate cancer cells. The androgen receptor and RNase L interact in a ligand dependent manner, which may be associated with prostate cancer initiation and progression [47]. Furthermore, overexpression of wild type RNase L conferred IFN sensitivity to a dihydrotestosterone inducible reporter gene, whereas R462Q-mutated RNase L is unable. It is suggested that

RNASEL variants Glu265X and Arg462Gln might contribute to the tumorigenesis of sporadic and familial pancreatic cancer [48]. Taken together, RNase L can be considered as a potent tumor suppressor although the molecular mechanism remains to be elucidated.

1.1.3.5 The involvement of RNase L in the immune system

In addition to its role in antiviral infection, apoptosis and anti-proliferation, RNase

L may also be involved in the immune system because tissue distribution analysis has revealed that RNase L is highly expressed in the thymus, spleen and most of immune cells

[49]. Recent genetic and biological studies suggest that RNase L may be a potent regulator

15 in modulating the immune response to exogenous pathogens and endogenous malignancies

[2]. The impact of RNase L on the regulation of the thymus gland and subclasses population of T cells is observed. It also has been shown that skin allograft was suppressed in mice lacking RNase L, implicating the involvement of RNase L in T cell immunity. In addition,

RNase L also exhibits extraordinary immunomodulatory ability in modifying a number of cytokine secretions in immune cells by which it mediates biological activities [49]. As critical factors in the B cell directed humoral immunity, these cytokines strictly control the initiation of immune response, the antibody class switching process and the populations of immunoglobin isotypes, acting as immunological switches against specific antigens.

Indeed, a cancer vaccine study had reported that alphavirus-based DNA vaccination against a non-mutated tumor-associate self-antigen (tyrosinase related protein-1, TRP-1) was severely impaired in RNase L deficient mice, indicating the involvement of RNase L in B cell mediated immunity [50]. In clinical trials, RNase L has been recognized for many years as a marker for diagnosing chronic fatigue syndrome (CFS), an illness associated immunological abnormalities with unknown etiology [2]. All these findings highlight the potential role of RNase L in the immune system. A schematic of the 2-5A/RNase L system, is shown in Figure 1.6 [15]

Figure 1.6 The 2–5A/RNase L pathway

(Bisbal C, Silverman RH. Biochimie. 2007)

1.1.3.6 RNase L affects lipid metabolism

RNase L was ubiquitously expressed in all of the tissues, not only in the immunological tissues, but also in the metabolic tissues, such as the adipose tissues and skeletal muscles [51,52]. The relationship between RNase L and human disease is rarely known although it may participate in a variety of pathological processes. The role of RNase

L in metabolic disorders had been investigated by using mouse embryo fibroblasts (MEFs).

RNase L affected differentiation of adipocyte through regulating the expression of CHOP-

10, a negative regulator of adipogenesis [52, 53]. RNase L knockout mice had large adipose tissues that may be caused by adipocyte hyperplasia. Ectopic fat deposition in liver and kidney was observed in RNase L deficient mice. Adipocyte differentiation and lipid accumulation was reduced by up regulation of Prep-1, an adipogenesis inhibitor, in RNase

L knockdown 3T3-L1 cell line [54]. The reduced RNase L levels in human serum may result in hypo-adiponectinemia in adipose tissue, which is capable of mediating the metabolic effects on peripheral metabolic tissues. The serum RNase L levels were found to be inversely associated with metabolic syndrome (MetS), unfavorable metabolic profiles, and age [55].

1.2 Overview of Diabetes Mellitus (DM)

Diabetes mellitus is a group of chronic metabolic diseases characterized by hyperglycemia resulting from insulin resistance, inadequate insulin secretion, or excessive glucagon secretion [56]. Manifestations of Diabetes include hyperglycemia and the classic symptoms of diabetes are glycosuria, polyuria, polydipsia, polyphagia and weight loss.

18 Diabetes mellitus is classified into two types: type I and type II, based on their pathogenesis [57]. Type I diabetes is caused by absolute deficiency of insulin secretion and type II diabetes is due to the resistance in insulin action and lack of an inadequate compensatory insulin secretory response. According to the American Center for Disease

Control, 9.4% of the population, 30.3 million people, have type I or II diabetes, and 1.6 million new cases of diabetes were diagnosed in people aged 20 years or older in 2017.

The number of adults living with diabetes has almost quadrupled since 1980 to 422 million adults. This dramatic rise is largely due to the rise in type 2 diabetes as a result of increased population in overweight and obesity.

1.2.1 Type I Diabetes Mellitus

Type I Diabetes Mellitus (T1DM) which accounts for less than 10% of those with diabetes, also known as juvenile diabetes, insulin-dependent diabetes or autoimmune diabetes, usually happens in people younger than 30, even though it can occur at any age[58]. It is a chronic autoimmune disorder caused in genetically susceptible individuals by environmental factors without a family history [59]. T1DM results from the cellular- mediated autoimmune destruction of β-cells in the pancreas, leading to insulin deficiency and hyperglycemia.

Only about 10–15% of the patients have a first- or second-degree relative with the disease, implicating it may not be genetically associated. However, the risk for developing

T1DM in the lifetime is significantly increased in relatives of patients. About 6% of children, 50% of monozygotic twins and 5% of siblings present the disease compared to

0.4% prevalence of the general population [60].

19 1.2.2 Type II Diabetes Mellitus

Type II Diabetes Mellitus (T2DM) accounts for 90–95% of those with diabetes, previously named as non-insulin dependent diabetes or adult-onset diabetes [57]. T2DM is a complex heterogeneous group of metabolic conditions characterized by increased levels of blood glucose and caused by impairment in both insulin action and insulin secretion [61].

Although the clear pathogenesis is not known, autoimmune destruction of β-cells does not occur, and patients do not have any of the other causes of diabetes. Current explanation of the pathogenesis of T2DM includes a defect in insulin-mediated glucose uptake in muscle, a disruption of secretory function of adipocytes, an impaired insulin action in liver, and a dysfunction of the pancreatic β-cells [62]. T2DM may result in severe complications, including renal failure, blindness, slow healing wounds, and arterial diseases.

In North American, about 90–95% of cases of diabetes are type II diabetes and nearly 20% of the population over the age of 65 has T2DM [63]. In many countries over the world, 5–10% of the total health care budget have been used for T2DM cares and treatment.

1.2.3 Obesity and Type II Diabetes

1.2.3.1 Overview of Obesity

The definition of overweight and obesity is an excess accumulation of adipose tissue to an extent that impairs both physical and psychosocial health and well-being [64].

Body mass index (BMI) is a measurement obtained by dividing a person's weight by the square of the person's height and its normal range is 25–30 kg/m2. When BMI is over 30 kg/m2, people are generally considered as obesity [65].

20 Obesity currently affects more than 600 million people worldwide and is associated with more than 45 complications [66]. Approximately 60%-70% of the adult population in the US is considered to be overweight or obese. Similar trends are being noticed worldwide

[67]. The increased prevalence of obesity these days has drawn attention to the world.

Obesity is linked to many medical, psychological, and social conditions such as cardiovascular diseases, type II diabetes, obstructive sleep apnea, certain types of cancer, osteoarthritis and asthma [68,69].

1.2.3.2 Linking of Obesity to Diabetes

It is estimated that about 90% of T2DM is attributable to excess weight [70]. The increased prevalence of type 2 diabetes is likely linked to the rise of obesity. However, obesity is not a requisite or sufficient condition for T2DM development. The connection between obesity and diabetes in pathophysiology is mainly attributed to two factors: insulin resistance and insulin deficiency [71]. Indeed, the majority of obese population do not develop T2DM, but only individuals who are genetically predisposed develop type II diabetes. Nonetheless, studies have shown that obesity plays a direct role in the pathogenesis of diabetes and listed obesity as one of the major risk factors for this disease

[72].

1.3 Insulin signaling pathway

Insulin is a hormone secreted by β-cells in the pancreatic islets. The function of insulin is to maintain the blood glucose at a normal level by facilitating cellular glucose uptake, regulating carbohydrate, protein and lipid metabolism and promoting cell division

21 and growth through its mitogenic effects. Insulin activates a variety of biological processes by acting on the tyrosine kinase receptors on the cell membrane. Activation of the insulin receptor initiates a series of phosphorylation events that lead to the activation of downstream important enzymes that control metabolism and growth.

The insulin signaling pathway is precisely regulated, both positively and negatively, to ensure proper signal duration and intensity (Figure 1.7). Binding of insulin to its receptor, results in the autophosphorylation of the insulin receptor (IR) on its tyrosine residues, followed by tyrosine phosphorylation of insulin receptor substrates (IRS). This allows IRSs to associate with SRC homology 2 (SH2) domains of the regulatory subunit of phosphoinositide 3-kinase (PI3K). Once PI3K is activated, the catalytic subunit phosphorylates phosphoinositides at the 3' position of the inositol ring or proteins at serine residues. PI3K activates PtdIns(3,4)P2/PtdIns(3,4,5) P3-dependent kinase 1 (PDK1), followed by activation of a serine kinase PKB/Akt. Activation of PKB results in the translocation of GLUT4 vesicles from intracellular to the plasma membrane, where

GLUT4 can uptake of glucose into the cell. PKB also activates mTOR-mediated activation of protein synthesis by PHAS/elf4 and p70s6k [73-76].

Figure 1.7 Insulin-signaling pathways

(Boucher J, Kleinridders A, Kahn CR. Cold Spring Harb Perspect Biol. 2014)

23 1.3.1 Insulin Receptor

Insulin mediates their biological effects through the insulin receptor (IR), which belongs to the receptor tyrosine kinase (RTK) family and acts as an extracellular alkali sensor [77]. The insulin receptor is a tetrameric protein which consists of two extracellular subunits insulin receptor-α and two transmembrane subunits insulin receptor-β (Figure 1.8).

Both subunits generated from a single precursor by proteolytic cleavage are linked together through the disulfide bonds [78]. IR is an allosteric enzyme in which the α-subunit inhibits the tyrosine kinase activity of the β-subunits. Binding of insulin to the α-subunit leads to the dimerization of the receptor in the plasma membrane and the autophosphorylation of the β-subunit at Tyr1158, Try1162/1163, which is the first step of the activation of the insulin signaling pathway. The activation of IR results in the phosphorylation of several downstream substrates, such as IRS1, SHC and Grb-2-associated protein (GAB1), leading to the activation of the Ras/MAPKs and PI3K/Akt pathways [79].

Figure 1.8 Insulin Receptor

(https://www.azuravesta.com/bhsc)

25 1.3.2 Insulin Receptor Substrate

Insulin Receptor Substrate (IRS) proteins are a family of cytoplasmic adaptor molecules involved in transducing extracellular signals from receptors to downstream proteins in the insulin signaling pathway [80,81]. The members of the IRS family are referred to as IRS-1 through IRS-6. IRS-1 and IRS-2 are the first two family members identified and are ubiquitously expressed as the primary mediators of insulin dependent mitogenesis and regulation of glucose metabolism in most types of cells. IRS-1 mediates insulin signaling mainly in the skeletal muscle and IRS-2 functions more in the liver, adipose tissue and β-cells of the pancreas [82]. IRS-3 is expressed in only rodents [83,84], while IRS-4 is found only in human brain, kidney, thymus and liver [85]. IRS-5 and IRS-

6, also named as DOK4 and DOK5, have limited tissue expression and are distantly related to IR signaling [86-88].

IRS proteins are phosphorylated by the activated insulin receptors on multiple tyrosine residues (Figure 1.9), which form the binding sites for the intracellular molecules containing Src-homology 2 (SH2) domains [89]. The binding of SH2-proteins to IRS proteins serves several different purposes. Once IRS proteins are phosphorylated, they bind to the SH2 domain proteins such as the subunit of PI3K, the tyrosine protein phosphatase

SHPTP-2, and growth factor receptor binding protein Grb-2 [90]. The activation of those

SH2 domain proteins initiates the signaling cascades, leading to the activation of a variety of downstream signaling pathways related to metabolic responses, cell survival, cell growth and cell differentiation.

Figure 1.9 Binding site of IRS proteins

(Lee YH, White MF. Arch Pharm Res. 2004)

27 1.3.3 Phosphatidylinositol-3 kinase

The Phosphatidylinositol-4,5-bisphosphate 3-kinase, also known as phosphoinositide 3-kinases (PI3Ks), are a family of lipid enzymes catalyze the addition of phosphate on the D3 position of the inositol ring of phospoinositol on the cell membrane, leading to the generation of PI 3-phosphate [91,92]. The enzyme consists of an 85 kDa regulatory subunit containing two SH2 domains and existing in several isoforms (p85-α, p85-β, p55/AS53, p55PIK, and p50), and a catalytic 110 kDa subunit having as α, β, γ, and

δ isoforms [93]. 3-Phosphorylated inositides function as intracellular messengers to activate PI-dependent kinases and subsequently impact cell growth, proliferation, differentiation, motility, survival and intracellular trafficking [94].

The PI 3-kinase is one of the IRS (IRS-1 and IRS-2) targets, which is able to convert

PIP2 to PIP3 by phosphorylation of specific phosphoinositides and is the first SH2-protein found to associate with IRS-1 [80]. Both SH2 domains in p85 and p55PIK interact specifically with phosphorylated IRS-1 in the intact cells and this event is demonstrated with recombinant IRS-1 in vitro. IRS-1 contains at least four sites that can interact with the

SH2 domains of p85 subunit [89]. Tyrosine phosphorylated IRS-1 activates the PI-3 kinase through interacting with p85 [95, 96]. Activation of PI 3-kinase is important for a variety of insulin actions, including activation of the downstream kinases such as phosphoinositide-dependent kinase-1 (PDK1) and AKT [97]. Blocking PI 3-kinase with wortmannin, a fungal inhibitor, inhibits insulin-stimulated glucose uptake [98, 99], glycogen, lipid, and protein synthesis [100-103], and gene expression [104, 105].

28 1.3.4 PKB/AKT pathway

The protein kinase B (PKB), also known as Akt, is a serine/threonine protein kinase that plays an important role in diverse signaling cascades downstream of growth factor receptor tyrosine kinases, involved in cell survival, growth, migration, proliferation, polarity, lipid and glucose metabolism, cell cycle progression, muscle and cardiomyocyte contractility, angiogenesis, and self-renewal of stem cells [106].

In the insulin signaling pathway, Akt is activated by insulin or insulin-like growth factor through the PI3K dependent cascade [107]. In the absence of insulin stimulation, all three isoforms of the Akt (Akt1/2/3) kinase are inactive. Full activation of Akt is a multistep process. PI3K is able to phosphorylate two key phosphorylation sites on Akt1 (Figure 1.10) at T308 in the activation, or T-loop, of the catalytic protein kinase core, and S473 in a C- terminal hydrophobic motif [108], via PDK1 and PDK2 respectively [109]. PDK (3- phosphoinositide-dependent protein kinase) is the major upstream kinase responsible for the phosphorylation and activation of Akt, regulated by PI3K [110]. PDK-1 contains a PH domain that binds to membrane-bound PIP3 that stimulates the PDK-1 activation. PDK-1 phosphorylates and activates Akt at Thr-308 residue [111]. However, Akt phosphorylation at Ser-473 requires a fully activation which is accomplished by the mammalian target of rapamycin complex 2 (mTORC2) [112].

Figure 1.10 Molecular Mechanisms of AKT Regulation

(Manning BD, Toker A. Cell. 2017)

30 Activation of Akt by PDK1 and mTORC2 stimulates the phosphorylation and activation of a series of downstream targets. Akt phosphorylates tuberous sclerosis complex protein 2 (TSC-2), inducing the degradation of the tumor suppressor complex that consists of TSC-2 and TSC-1, followed by the activation of mTORC1. The mTORC1 complex inhibits 4E binding protein 1 (4E-BP1) by phosphorylation of 4E-BP1 and activates ribosomal protein S6 kinase (S6K), leading to the regulation of lipid metabolism, protein synthesis, and cell growth [113].

Figure 1.11 Schema of the PI3K/AKT pathway

(Mayer IA and Arteaga CL. Annu Rev Med. 2016)

32 1.3.5 Glucose transporter

GLUT family mediates the transport of monosaccharides, polyols, and other small carbon compounds across the membrane of cells in the body. GLUT proteins are a group of integral membrane proteins encoded by SLC2 genes [114,115]. There are 14 mammalian glucose transporters and they belong to the sugar porter family of major facilitator superfamily (MFS) [116,117]. Based on their sequence similarity, the 14 GLUTs can be categorized into three classes: Class I (GLUT1–4 and14); Class II (GLUT5, 7, 9 and 11); and Class III (GLUT6, 8, 10, and 12) [118].

Among these GLUTs, only GLUT4 and GLUT12 are insulin dependent glucose transporters [119,120]. GLUT4 is expressed mainly in insulin-responsive tissues such as adipose tissue, skeletal muscle, and cardiomyocytes, where it mediates glucose uptake in response to acute insulin stimulation [121]. Although GLUT4 is the predominant glucose transporter in insulin-sensitive tissues, recently there are evidences showing that GLUT12 could be a novel insulin dependent transporter in GLUT family. However, its physiological role has not been clearly elucidated [122].

The translocation of GLUT4 from the intracellular site to the surface of the cell where transporting glucose occurs is stimulated by the activation of Akt [123]. As the upstream component of Akt, PI3K is crucial in the insulin signal transduction and indispensable for the effects of insulin on GLUT4 translocation and glucose uptake [124].

In type II diabetic patients, the activity of PI3K in the skeletal muscle is reduced, indicating that PI3K affects the insulin signal transduction and probably contributes to the impaired translocation of GLUT4 and insulin resistance.

33 1.4 The Physiological Effects of Insulin

Insulin is the essential hormone regulates cellular energy supply and macronutrient balance, directing anabolic processes in our body. Insulin is crucial for the intra-cellular transport of glucose into insulin-dependent tissues such as muscle, liver and adipose tissue

[125].

1.4.1 Liver

Insulin stimulation in the hepatocyte of liver leads to the suppression of gluconeogenesis and glycogenolysis, and increases glycogen synthesis, results in decreasing of glucose output from the liver [126]. This effect is a directly inhibition through phosphoenolpyruvate carboxykinase (PEPCK) and the inhibition of lipolysis in adipose tissue followed by the reduction of glycerol as a substrate for gluconeogenesis [127].

Insulin also increases the synthesis of fatty acid, predominantly palmitic acid, and cholesterol by activating SREBP-c [128]. Hepatocyte insulin resistance results in increased glucose output and dyslipidemia.

1.4.2 Skeletal Muscle

The major target tissue for insulin–stimulated glucose disposal is skeletal muscle.

Insulin stimulates skeletal muscle to take up glucose through an increase of GLUT4 translocation to the plasma membrane [129]. About 80% of the glucose is taken up by skeletal muscle in normal people. Insulin also stimulates the glycogen synthesis in skeletal muscle by activating glycogen synthase (GS) [130]. Phosphorylation of Glycogen synthase kinase 3 (GSK3) inhibits its kinase activity and decrease the phosphorylation of GS, results

34 in the increase of glycogens synthase activity [131,132]. Furthermore, insulin also stimulates other processes in muscle such as the activation of the mammalian target of rapamycin (mTOR) pathway [133].

1.4.3 Adipose Tissue

Insulin stimulates glucose uptake through the GLUT4 dependent pathway in adipose tissue [134]. Adipose tissue uses glycolysis for the energy purposes. Insulin promotes re-esterification of fatty acids into triglycerides [135,136].

1.5 Insulin Resistance

The definition of insulin resistance is a subnormal biologic response to a given concentration of insulin [137]. It is a common feature of several disorders such as obesity, nonalcoholic fatty liver disease (NAFLD), atherosclerosis, and type II diabetes (T2DM).

Physiologically, the action of insulin is influenced by the interplay of other hormones in the whole body. Excess secretion of other hormones such as IGF-1, glucagon, glucocorticoids and catecholamines may contribute to insulin resistance, but does not account for the majority of insulin resistant states.

Insulin resistance in most cases is believed to be manifest at the cellular level via post-receptor defects in insulin signaling. Although findings in experimental animals with respect to a range of insulin signaling defects, their relevance to human insulin resistance is presently not clear. Some of the possible mechanisms including down-regulation of tyrosine phosphorylation and deficiencies or genetic polymorphisms in the insulin receptor,

35 IRS proteins or PIP-3 kinase, as well as abnormalities of the GLUT 4 function have been reported [138].

The link between insulin resistance and type II diabetes has been well established.

Insulin resistance is not only the most powerful predictor of future development of type II diabetes, but also a therapeutic target for this disease [139].

1.6 Rationale for the Studies

Although insulin resistance has been rather extensively studied, the molecular pathogenesis and mechanisms leading to the disorder remain vague. The previous study in our lab showed that RNase L might play an important role in the metabolic pathways of mice fed with a high-fat diet. The levels of glucose, cholesterol, and triglyceride were significantly higher in the plasma from RNase L-/- mice compared with that from RNase

L+/+ mice (Fig. 1.12), while the level of insulin was also higher in the plasma from RNase

L-/- mice. The observations suggest that RNase L may be involved directly or indirectly the metabolic pathways through regulating insulin sensitivity [140].

Figure 1.12 Effect of RNase L on diet-induced obesity

(Zeng C, et al. J Endocrinol. 2014)

37 In this study, we determined the mechanistic basis of RNase L function in insulin resistance through its role in mediating the insulin signaling pathway. This will greatly expand our understanding of the basic cellular function of RNase L and its role in diabetes, particularly in carbohydrate metabolism. Our results will provide new evidence highlighting the role of RNase L, an enzyme well known to be associated with host defense against viral infection, in metabolic diseases, such as diabetes. Therefore, novel therapies for treatment and prevention of the disease could be designed based on the selective regulation and inhibition of RNase L.

1.7 Specific Aims and Hypotheses

In this project, we hypothesize that RNase L plays an important role in insulin signaling through regulating expression and activation of certain components in the pathway, leading to the regulation of insulin sensitivity.

Specific aim 1: Determine the role of RNase L in insulin signaling and sensitivity.

Specific aim 2: Investigate the mechanism by which RNase L regulates the insulin signaling pathway.

CHAPTER II

THE ROLE OF RNASE L IN INSULIN SIGNALING PATHWAY

2.1 Introduction

Type II diabetes mellitus (T2DM) is characterized by dysregulation of carbohydrate, lipid and protein metabolism, due to impaired insulin secretion and/or insulin resistance.

[141]. Binding of insulin to its receptor activates insulin receptor and a family of insulin receptor substrates (IRSs), especially IRS1 and IRS2 [142]. The phosphorylated IRS proteins bind and activate intracellular signaling molecules PI3K. PI3K promotes GLUT4 translocation to the plasma membrane, resulting in glucose uptake into skeletal muscle and adipose tissue.

Insulin resistance in obesity and T2DM has been linked to the PI3K pathway

[143,144]. Insulin resistance is usually associated with phosphorylation of IRS proteins, which inhibits tyrosine phosphorylation, leading to the disorder [145]. In some cases, phosphorylation of IRS also increases its degradation, further contributing to the insulin resistance [146].

RNase L, an IFN-inducible enzyme, plays an important role in IFN functions against viral infection and cell proliferation [1]. Previous data obtained by our laboratory

39 have shown that: 1) aged RNase L knockout mice had heavier body weight and fat mass than wild type mice (Fig. 2.1); 2) The levels of glucose, cholesterol, and triglyceride were respectively higher in the plasma from RNase L knockout mice, while the level of insulin in the plasma was also higher (Figure 1.12); 3) RNase L mediates the activation of the

MEK/ERK and PI-3K/Akt pathways (Fig. 2.2); and 4) p70S6K, a serine kinase downstream in the mTOR pathway, is activated in RNase L knockout mouse embryonic fibroblasts (MEFs), especially stimulated with insulin (Fig. 2.3).

However, a direct link between RNase L and insulin resistance has yet to be clearly established. In this project, we found that RNase L plays an important role in regulating the expression of certain gene products which mediates the insulin signaling pathway and insulin sensitivity. Our long-term goal is to develop RNaseL-based novel strategies to prevent and/or treat diabetes.

Figure 2.1 Involvement of RNase L in lipid metabolism

(Fabre O, et al., Cell Death Differ. 2012)

Figure 2.2 RNase L mediates activation of ERK and AKT

Primary RNase L+/+ and -/- MEFs were treated with 1μg/ml of LPS (A) or 100 nM insulin (B) for various times. Activation of ERK and AKT was determined by Western blot with antibodies for p-ERK/ERK2 (Santa Cruz) and p-Akt/ Akt (Cell Signaling).

Figure 2.3 Effect of RNase L on phosphorylation of p70s6k

RNase L +/+ and -/- MEFs were treated with 100 nM insulin (Sigma) for various times. The cell extracts were subjected to Western blot analysis with antibodies for p-p70S6K (Cell Signaling) and β-Actin (Santa Cruz).

43 2.2 Materials and methods

2.2.1 Experimental animals

We maintained two breeding colonies for this study: normal C57BL/6 mice (RNase

L +/+) and C57BL/6 RNase L knock out mice (RNase L -/-). RNase L-/- C57BL/6 mice (a generous gift from Dr Robert Silverman, Cleveland Clinic) were generated with a targeted disruption of the RNase L gene as described [147].

We housed 2 females and 1 male (maximum of 3 adults) per breeding cage, and separate females before pups are born to keep1 litter per breeding cage. If females had 2nd litter, older litter was weaned immediately. Females were paired for breeding at 5-6 weeks or older and allowed to breed to a maximum of 9 months of age. Males were bred as early as 10 weeks and were retired at 12 months.

RNase L is an antiviral protein and mice deficient RNase L may be more vulnerable to pathogens. Aseptic microisolator techniques were used for experimental manipulations and cage changes. All mice were housed four per polypropylene cages (25 x 16 x 13 cm) with abundant pine bedding in a temperature of 25°C under a 12-hr dark/light cycle. Sterile microisolators, autoclaved food and water were used for the mice.

Animals were maintained and used in strict accordance with the guidelines of the

Institutional Animal Care and Use Committee (IACUC) of Cleveland State University. The experimental procedures were approved by the Committee on the Ethics of Animal

Experiments of Cleveland State University with an approval number of 21111-ZHO-AS.

All efforts were made to reduce suffering. Mice were euthanized under CO2 for 5 min and then their necks were dislocated at the end of the experiments.

44 2.2.2 Experimental cell lines

2.2.2.1 Primary Mouse embryo fibroblasts (MEFs)

Fibroblasts are a group of heterogeneous resident cells of mesenchymal origin that have various locations, diverse appearances and distinctive activities. Primary Mouse

Embryonic Fibroblasts are a type of fibroblast isolated directly from mouse embryos.

Culturing of mouse MEF cells represents a powerful tool to test gene function due to their easy accessibility, rapid growth rates, and the possibility of a large number of experiments.

In this project, MEFs were isolated from RNase L wild type and deficient mice and used to investigate the role of RNase L in the insulin signaling pathway.

2.2.2.2 BC 10 T-ag cell line

Primary cells only undergo a predetermined and finite number of cell divisions in culture [148]. After amplification in limited passages, primary cells enter a state where they can no longer divide. There are several different methods for immortalizing mammalian cells in culture conditions. One of the methods is to transfect the cells with an oncogene or a viral gene, such as the simian virus 40 (SV40) T antigen, to convert them as immortalization [149]. SV40 T antigen is a simple and reliable agent for the cell immortalization, especially the mechanism of SV40 T antigen in immortalization is well established [150].

In this project, T-antigen immortalized MEFs of RNase L +/+ and RNase L -/-, generated previously in our lab, were used for the investigation of insulin signaling pathway.

45 2.2.2.3 SK-HEP-1cell line

In order to determine whether RNase L medias the insulin signaling pathway and is associated with insulin resistance in hepatocytes, we used SK-HEP-1 hepatocytes, a liver adenocarcinoma cell line (ATCC), analyzed the expression and insulin-stimulated phosphorylation of insulin signaling intermediates. The CRISPR/Cas9 method was used to generate an RNase L knockout SK-HEP-1 cell line.

2.2.3 Isolation of primary Mouse embryo fibroblasts

Five embryos from each type of the female mice (RNase L+/+ and RNase L-/- mice) were harvested aseptically in phosphate-buffered saline (PBS: 140mM NaCl, 2mM KCl,

8mM Na2HPO4, 1.5mM KH2PO4 (pH 7.4), Cleveland Clinic, Cleveland, Ohio) at the stage of 16 days after the appearance of the copulation plug. The limbs of each embryo were cut and chipped to very small pieces of 1-2 mm and then transferred to a 15 mL centrifuge tube. Trypsin–EDTA solution (5 mL, 0.05%) was added to each tube and incubated at 37°C for half an hour and then in 4°C overnight for the further digestion. The cells were resuspended in 10 mL of RPMI 1640 medium containing 10% (V/V) of fetal bovine serum (FBS). The cells were then plated in tissue culture dishes to be amplified for use in the experiments.

2.2.4 Isolation of primary mouse hepatocytes

Liver tissues were collected from RNase L+/+ and RNase L-/- male mice. The release of hepatocytes from the liver was performed by pushing the tissue with a syringe insert against the filter of a cell strainer (70 μm pore size) in a petri dish containing 10 mL of

46 RPMI 1640 medium. After the centrifuge, the supernatant was discarded, and the cells were resuspended with 5 mL of the Red Blood Cell Lysis Buffer (Sigma-Aldrich, St. Louis, MO).

The cell suspension was placed on the ice for 5 min and was centrifuged again to remove the lysis buffer. The cell pellet was re-suspended in the normal cell culture medium, transferred to a 100mm cell culture dish for the experiment.

2.2.5 RNase L knockout in SK-Hep-1 cell line

2.2.5.1 Introduction of the CRISPR/Cas 9 method

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) genes are found in a large number of prokaryotes. The primary function of CRISPR and Cas is to recognize and destroy foreign nucleic acids such as plasmids and viruses, providing the host with an adaptive immune system. The CRISPR/Cas9 system has been harnessed to create a simple, RNA-programmable method to mediate genome editing in mammalian cells. In recent years, this system has been widely used as a tool for gene knockout or knockin. To create gene disruptions (Figure 2.4), a single guide RNA

(sgRNA) is generated to direct the Cas9 nuclease to a specific genomic location. Cas9- induced double strand breaks are repaired via the non-homologous end joining (NHEJ)

DNA repair pathway. The repair is error-prone, and thus insertions and deletions may be introduced to disrupt gene function.

Figure 2.4 The principle of CRISPR/Cas9-mediated gene disruption

(https://www.takarabio.com/learning-centers/gene-function/gene-editing/gene-editing-

technology-overviews/introduction-to-the-crispr/cas9-system)

A single guide RNA (sgRNA), consisting of a crRNA sequence that is specific to the DNA target, and a tracrRNA sequence that interacts with the Cas9 protein (A), binds to a recombinant form of Cas9 protein that has DNA endonuclease activity (B). The resulting complex will cause target-specific double-stranded DNA cleavage (C). The cleavage site will be repaired by the nonhomologous end joining (NHEJ) DNA repair pathway, an error- prone process that may result in insertions/deletions (INDELs) that may disrupt gene function (D).

48 2.2.5.2 RNase L gene knockout

SK-HEP-1 cells were seeded in 6-well plates and cultured for one day to reach a

70% confluence. For each of the transfection (a single well in 6-well plates), 1μg of RNase

L CRISPR/Cas9 KO plasmid and RNase L HDR plasmid (Santa Cruz Biotechnology, TX) were mixed with transfection reagent containing polycationic lipids which facilitate DNA delivery into cells, and the mixture was incubated for 20 min in the room temperature. The mixture was then added to the cells and incubated for 48 hours in 37°C. After two days, the transfected cells were transferred to a 100mm cell culture dish and selected by puromycin (Santa Cruz Biotechnology, TX) at a final concentration of 1μg/mL. The selection was continued for two weeks and the media was replaced with fresh one containing 1μg/mL of puromycin every 2 days. Then the clones were selected by using limiting dilution cloning (LDC) in 96‑well plates [163]. The absence of RNase L expression was then verified by Western blot.

2.2.6 Cell culture and insulin treatment

RNase L +/+ and -/- Primary MEFs, BC10 T-ag MEFs, SK-HEP-1 cells and primary hepatocytes were maintained in Roswell Park Memorial Institute 1640 (RPMI 1640, with

L-glutamine) Media (Cleveland Clinic, Cleveland, Ohio) supplemented with 10% (V/V) fetal bovine serum (FBS, Atlanta Biologicals, Inc., Flowery Branch, Georgia), 100U/ml penicillin-streptomycin.

In the experiments, cells were starved in serum free media for 24 hours prior to insulin stimulation. Insulin was diluted from stock solutions in PBS without additives and added to cells at a final concentration of 100 nM for 10, 30, 60 min at 37°C. Unstimulated

49 cells were served as controls. After the treatment, cells were rinsed and harvested with ice- cold Tris-buffered saline (TBS). After the centrifuge, the cell pellets were freeze on dry ice and stored in -80°C for the further analysis.

2.2.7 Sample preparation

Proteins were extracted by using Triton X-100 lysis buffer (50mM Tris-HCl (pH

7.4), 150mM NaCl, 1% Triton X-100, 5mM EDTA, 50mM β-glycerophosphate) with freshly added a protease inhibitor cocktail (Calbiochem, CA) and a phosphatase inhibitor cocktail (Thermo Fisher Scientific, Inc.). Lysis buffer was added to the cell pellets by 2X of the pellet volume. After votexing and sonication, the cell lysate was centrifuged at

12,000 rpm 4°C for 15 minutes. The supernatant was transferred to a new 1.7 mL centrifuge tube for the western blot analysis.

2.2.8 Western blot analysis

Western blot experiments were performed by using SDS-PAGE and wet transfer in this project. Protein samples were separated by 8% SDS-PAGE gel and thereafter transferred onto PVDF membrane (Thermo Fisher Scientific Inc). After transfer, the membrane was blocked in TBST containing 5% BSA for one hour and then incubated with specific first antibody (1:2000 diluted in blocking buffer) listed in Table 2.1 overnight at

4°C. HRP-conjugated second antibody (1:3000 diluted in blocking buffer) was used and incubated at room temperature for 1hour. The membrane was developed in an ECL2 plus reagent (Thermo Fisher Scientific Inc) and then exposed to x-ray film.

50 Antibody Isotype Catalog NO. Company

β-Actin Mouse sc-47778 Santa Cruz

β Tubulin Mouse sc-5274 Santa Cruz

GAPDH Mouse sc-32233 Santa Cruz

Insulin Receptor-α Mouse sc-710 Santa Cruz

Insulin Receptor-β Mouse sc-57342 Santa Cruz

p-Akt (Thr308) Rabbit #4056 Cell Signaling

P-Akt (Ser473) Rabbit #9271 Cell Signaling

p-IRβ (Tyr1150) Mouse sc-81500 Santa Cruz

p-IRβ (Tyr1162) Rabbit sc-25103 Santa Cruz

p-IRS-1 (Ser302) Rabbit #2384 Cell Signaling

p-IRS-1 (Ser307) Rabbit sc-33956 Santa Cruz

p-IRS-1 (Tyr895) Rabbit #3070 Cell Signaling

p-IRS-1 (Tyr1222) Rabbit #3066 Cell Signaling

p-p70S6K (Thr389) Rabbit #9206 Cell Signaling

p-VCP Rabbit SAB4504738 Sigma

Ubiquitin Mouse sc-8017 Santa Cruz

VCP Mouse sc-20799 Santa Cruz

Table 2.1 Antibodies I

51 2.3 Results

2.3.1 Deficiency of RNase L attenuates the insulin signaling pathway in MEFs

To determine if RNase L has any effects on the activation of the insulin signaling pathway in fibroblast, RNase +/+ and -/- primary MEFs and immortalized MEFs BC10 T-ag cells were cultured in serum-free medium for 24 hours and then stimulated with 200nM insulin for 0, 10, 30 and 60 min. Western blot analysis was performed to compare the activation of Akt and IRS between two types of cells (Figure 2.5). IRS-1 can be activated after the activation of Insulin Receptor [81]. Two phosphorylation sites of IRS-1at Ser 302 and Tyr 1222 were analyzed. As shown in Fig.2.5 A, both sites were phosphorylated upon insulin stimulation in RNase L +/+ cells. As one of the key enzymes in the insulin stimulated

PI3K pathway, Akt is activated by phosphorylated at its Thr308 residue [93]. Another residue of Akt at Ser473 can also be phosphorylated by the mTOR2 complex [112].

Interestingly, the phosphorylation level of Akt in RNase L deficient MEFs significantly decreased as shown in Figure 2.5 D. This observation was further confirmed by analyzing p-IRS (Ser 302) and p-Akt (Thr 308) in primary MEFs (Fig.2.6).

Figure 2.5 Effects of RNase L on activation of IRS-1 and Akt in MEFs

RNase L +/+ and -/- MEFs were treated with 200 nM insulin (Sigma) for various times. The cell extracts were subjected to Western blot analysis with antibodies for p-IRS-1(A), p-Akt (D) (Cell Signaling) and β Tubulin (Santa Cruz). Quantitative analyses were performed by using NIH image software for the ratios of p-IRS1 (ser302)/Tubulin (B), p-IRS1 (Tyr1222)/Tubulin (C), p-Akt (Thr308)/Tubulin (E) and p-Akt (Ser473)/Tubulin (F).

Figure 2.6 Effects of RNase L on activation of IRS-1 and Akt in primary MEFs

Primary RNase L +/+ and -/- MEFs were treated with 200 nM insulin (Sigma) for various times. The cell extracts were subjected to Western blot analysis with antibodies for p-Akt and p-IRS-1 (Cell Signaling) (A). Quantitative analyses were performed by using NIH image software for the ratios of p-IRS1 (ser302)/Tubulin (B) and p-Akt (Thr308)/Tubulin (C).

54 2.3.2 RNase L affects the expression and activation of Insulin Receptor in MEFs

Insulin Receptor is activated by insulin or insulin-like growth factor [73]. Since

RNase L attenuates the downstream of Insulin Receptor in MEFs, we examined the level by which RNase L affects the activation of insulin signaling. In these experiments, Western blot analysis was performed to determine the activation and expression of IR in RNase +/+ and -/- MEFs after insulin stimulation (Fig. 2.7). Diminished tyrosine phosphorylation at

Tyr 1150 and Tyr 1162 of IR was observed in the immortalized RNase L -/- MEFs (Fig. 2.7

A). The results were confirmed by using primary MEFs (Fig. 2.8). It has been well established that both α and β subunits are derived from a single-chain IR precursor through the proteolytic cleavage [162]. To determine if RNase L plays any role in the expression and the process of its proteolytic cleavage, the expression of IR subunit α and β was examined. The cells were treated with 200 nM insulin for 10, 30 and 60 min and the level of subunits were examined by Western blot analysis. The result has shown no difference in the levels of IR-α between wild-type and RNase L -/- cells, suggesting that RNase L may not significantly affect the binding of insulin. However, the level of IR-β significantly decreased in RNase L -/- MEFs (Fig. 2.7E), implicating that either RNase L may affect the cleavage of the IR precursor or IR-β may be vulnerable to be degraded, causing reduced activation of IR-β.

Figure 2.7 Effect of RNase L on the activation of IR in MEFs

RNase L +/+ and -/- MEFs were treated with 200 nM insulin (Sigma) for various times. The cell extracts were subjected to Western blot analysis with antibodies for IR (E) and p-IRβ (A) (Santa Cruz). Quantitative analyses were performed by using NIH image software for the ratios of p-IRβ (Tyr 1150)/β-Actin (B), p-IRβ (Tyr 1150)/β-Actin (C), IR Precursor/β Actin (F), IR α/β Actin (G) and IR β/β Actin (H).

Figure 2.8 Effect of RNase L on the activation of IR in primary MEFs

Primary RNase L +/+ and -/- MEFs were treated with 200 nM insulin (Sigma) for various times. The cell extracts were subjected to Western blot analysis with antibodies for p-IR β (Tyr 1150) (Santa Cruz) (A). Quantitative analyses were performed by using NIH image software for the ratios of p-IRβ (Tyr 1150)/β-Tubulin (B).

57 2.3.3 Effects of RNase L on Insulin Signaling in Primary Hepatocytes

Liver is one of the organs response to insulin stimulation. Insulin stimulates hepatocytes to suppress gluconeogenesis and glycogenolysis, and increases glycogen synthesis, resulting in increasing of glucose absorption by the liver [126]. To explore if

RNase L affects the insulin signaling in the liver, primary hepatocytes were isolated from the liver tissues of RNase L+/+ and RNase L-/- male mice as described in Reagents and

Methods. The cells were starved in serum-free medium for 2 hours before insulin treatment.

Hepatocytes were then treated with 200 nM insulin at different time points. The proteins in the cell extracts were separated by SDS-PAGE, followed by transferring them to a PVDF membrane for Western blot analysis. Similar to the results obtained by using MEFs, the levels of IR, p-IR, p-Akt in primary hepatocytes with or without RNase L upon insulin stimulation were significantly different as shown in Figure 2.9. In the RNase L deficient hepatocytes, the activation of IR was reduced after the insulin treatment, probably due to the downregulated level of IR-β. Furthermore, the phosphorylation of Akt at Thr 308 residue remarkably decreased, suggesting that deficiency of RNase L may inhibit the activation of the insulin signaling pathway by regulating the protein level and activation of

IR-β.

Figure 2.9 Effect of RNase L on Insulin Signaling in Primary Hepatocytes

Primary RNase L +/+ and -/- hepatocytes were treated with 200 nM insulin (Sigma) for various times. The cell extracts were subjected to Western blot analysis with antibodies for p-IRβ (Tyr 1150), p-Akt (Thr 308) (A) (Santa Cruz) and IR-β (D). Quantitative analyses were performed by using NIH image software for the ratios of p-IRβ (Tyr 1150)/Akt (B), p-Akt (Thr 308)/Akt (C) and IR-β/β-Tubulin (E).

59 2.3.4 RNase L mediates insulin signaling in SK-HEP-1 cells

To further investigate the effect of RNase L on insulin signaling in hepatocytes, we used CRISPR/Cas9 technology to knock out the RNase L gene in SK-HEP-1 cells, a human liver adenocarcinoma line. The cells were transfected with RNase L knockout plasmid and

HDR plasmid, and stable colonies were selected by using the medium containing 1 μg/mL puromycin for two weeks. The expression level of RNase L in each clone was determined by Western blot analysis with a monoclonal antibody to human RNase L. As shown in

Figure 2.10, the expression of RNase L in several clones has been completely knocked out.

To explore the impact of RNase L on the expression and activation of IR in human hepatocytes, RNase L WT and KO SK-HEP-1 cells were stimulated with 200nM of insulin after being starved for 24 hours in serum-free medium. As expected, deficiency of RNase

L decreased the insulin-stimulated phosphorylation of IR-β and IRS-1, its downstream target, because of the reduced protein level of IR-β (Fig. 2.11). The results were in accordance with the observations previously obtained from MEFs and primary hepatocytes.

Figure 2.10 Knockout of RNase L in liver cancer cells

The numbers indicate the clones selected by using puromycin after SK-HEP-1 cells were transfected with the CRISPR/Cas9 plasmid. The expression of RNase L in the cells were determined by Western blot analysis with an antibody against human RNase L (a gift from Dr. Robert Silverman, Cleveland Clinic)

Figure 2.11 RNase L mediates insulin signaling in SK-HEP-1 cells

RNase L +/+ and -/- SK-HEP-1 cells were treated with 200 nM insulin (Sigma) for various times. The cell extracts were subjected to Western blot analysis with antibodies for p-IRβ (Tyr 1150), p-IRS-1 (Ser 302), p-IRS-1 (Tyr 1222) (A) and IR β (E) (Santa Cruz). Quantitative analyses were performed by using NIH image software for the ratios of p-IRβ (Tyr 1150)/GAPDH (B), p-IRS-1 (Ser 302)/GAPDH (C), p-IRS-1 (Tyr 1222)/GAPDH (D) and IR-β/β-Actin (F).

62 2.3 Discussion

RNase L is expressed almost in all of the tissues including the liver, adipose tissues and skeletal muscles [51,52]. The levels of glucose, cholesterol, and triglyceride were significantly higher in the plasma from RNase L-/- mice fed with a high-fat diet, while the level of insulin was also notably higher than that in wild type mice under the same condition

[140], suggesting that RNase L may be involved directly or indirectly in the metabolic pathways through regulating insulin sensitivity. Although a direct effect of RNase L on insulin sensitivity has not previously been described, several lines of evidence present in our current study demonstrate a possible role for RNase L in the regulation of insulin signaling. In the experiments, we observed that IRS-1 and Akt, the key enzymes in the insulin signaling pathway, were down regulated after insulin stimulation in the RNase L deficient MEFs, primary mouse and human hepatocytes, which is due to the decreased expression and activation of IR-β.

Previously, the study in our lab revealed that RNase L was implicated in the onset of Type I diabetes. However, the main focusing in that study is to determine the effect of

RNase L on the immune response and β-cells destruction [140]. In the present project, it is the first time to demonstrate that RNase L contributes to insulin sensitivity.

Interestingly, the IR precursors in both RNase L +/+ and -/- cells are equally expressed, suggesting that RNase L may not affect at the mRNA level, but the process of proteolytic cleavage in the liver and muscle cells. The IR β-subunit is composed of an extracellular part (194 amino acids), a transmembrane domain (23 amino acids), and a cytoplasmic domain (403 amino acids) [161]. After binding to IR at the cell membrane, insulin activates the tyrosine-specific kinase of the intracellular domain of the IR β-subunit,

63 which subsequently phosphorylates the intracellular substrates. The reduced phosphorylation of IR-β in RNase L deficient cells indicated that the activation of IR decreased. Apparently, it was caused by the reduced protein level of IR β-subunit in RNase

L deficient cells. How RNase L mediating the cleavage process of the IR precursor remains largely unknown.

CHAPTER III

INVESTIGATE THE MECHANISM BY WHICH RNASE L REGULATES THE

INSULIN SIGNALING PATHWAY

3.1 Introduction

As stated in the background, RNase L is one of the key enzymes in the 2-5A system of IFN action against viral infection and in the control of cellular proliferation [1,2]. RNase L shows unique structural and functional motifs. The N-terminal half of the enzyme contains nine ankyrin repeats, highly conserved protein/protein interaction domains, and the 2-5A binding domain that is necessary for the activation of the enzyme [151]. The C-terminal half has a cysteine rich region, a protein kinase homology region and a ribonuclease domain responsible for RNA degradation (Figure 1.4).

Studies in insulin-resistant animal models and humans have clearly demonstrated that RNase L mediates insulin signaling via the IRS-1/ PI 3-kinase pathway and deficiency of RNase L resulted in reduced glucose uptake and utilization in insulin target tissues.

However, the nature of the target molecule (s) remains largely elusive. Interestingly, our studies showed that deficiency of RNase L in cells decreased the level of IR-β and attenuated the activation of the insulin signaling pathway. The equal expression of the IR

65 precursor suggests that RNase L may contribute to insulin sensitivity through regulating the proteolytic cleavage of the precursor protein. It has been well known that both α- and

β-subunits are derived from a single-chain precursor protein by cleavage, which then undergoes glycosylation, disulphide isomerization and dimerization in the endoplasmic reticulum (ER) [162]. Valosin-containing protein (VCP; p97; cdc48 in yeast) is a hexameric ATPase of the AAA family involved in multiple cellular functions, including degradation of proteins through the ubiquitin (Ub)-proteasome system (UPS). Previous studies in our lab showed that RNase L is associated with the phosphorylation of VCP. In this aim, we attempted to elucidate how RNase L mediates the insulin signaling pathway and identify the target molecules.

3.1.1 P97/VCP system

Endoplasmic-reticulum-associated protein degradation (ERAD)/proteasome pathway plays an important role in the quality control for newly synthesized proteins entering the secretory process. Nascent peptide chains fold improperly in the ER require retro-translocation into the cytosol where they are degraded by the proteasome, which is an energetically unfavorable event. A line of evidence demonstrate that p97VCP is involved in ERAD and other cellular functions such as membrane fusion, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and chromatin-associated processes [152-155]. P97/VCP utilizes the energy from ATP hydrolysis to extract its substrate proteins from cellular structures or multiprotein complexes in the ubiquitin signaling processes of the ERAD/proteasome pathway. A variety of P97/VCP associated factors have evolved for its function. Mutations or post-transitional modifications of this

66 protein have been shown to cause the multisystem proteinopathy associated with human diseases [156]. It has been well established that HMG-CoA reductase is a target of the

ERAD pathway in the presence of high levels of sterol as a feedback regulation of lipid homeostasis, in which the process is mediated by gp78-associated P97/VCP [157,158].

3.1.2 RNase L impacts phosphorylation of P97/VCP

Previously, we have reported that RNase L deficient mice display enlarged thymuses with substantially more cells in the cortex regions than that in the wild type thymuses [8], suggesting that RNase L may be involved in thymocyte proliferation and function. It is believed that protein tyrosine phosphorylation is an important event in the regulation of cellular proliferation and cell cycle progression in immune cells [159,160].

To determine the molecular targets of RNase L in the thymocytes, we examined the phosphorylated tyrosine protein (p-Tyr) profile in thymocytes isolated from the thymuses of RNase L+/+ and -/-mice by Western blot analysis probed with a p-Tyr antibody. Strikingly, the p-Tyr protein profiles were dramatically different in both types of thymocytes. Addition of ATP and P3A3, the activator of RNase L, did not significantly affect the p-Tyr protein profiles, suggesting that these p-Tyr proteins exist in the cells and they were not phosphorylated by any kinases in vitro. One of the major phosphorylated proteins was identified by using the proteomic analysis method as P97/VCP, a key regulator in the ubiquitin/proteasome pathway as mentioned above. The identity of p97/VCP was further confirmed by co-immunoprecipitation assays (Data not shown).

67 3.1.3 The ubiquitin–proteasome pathway and its impact on IRS-1

The ubiquitin–proteasome system is the major pathway of selective protein degradation in cells. The system degrades most short-lived cellular proteins and abnormal proteins. It has been demonstrated that the ubiquitin–proteasome system is connected to a variety of biological functions such as cell cycle control, apoptosis, and antigen presentation [167]. The degradation of proteins by the ubiquitin–proteasome system includes two steps. In the first step, the targeted proteins are marked by the covalent addition of the polypeptide ubiquitin by a complex containing ubiquitin-activating enzyme

(E1), ubiquitin-conjugating enzyme (E2) and ubiquitin-protein ligase (E3) [167-169]. Then the ubiquitin-conjugated proteins are recognized by the 26S proteasome, a proteolytic complex, subsequently degraded. It has been demonstrated that Serine/Threonine- phosphorylated IRS-1 proteins can be degraded by the proteasome system, which is regulated by the ratio of Ser/Thr versus Tyr phosphorylation of IRS-1[170,171], consequently affecting insulin signaling [172].

3.2 Materials and methods

3.2.1 Experimental cell line and cell culture

3.2.1.1 L929 PD12 cell line

To determine if the RNase enzymatic function of RNase L is necessary for its effect on the insulin signaling pathway, we over-expressed a dominant negative mutant (PD12) encoding 340 amino acids from 5’-terminal of the RNase L protein in L929 cells (a mouse fibroblast cell line). The mutant contains the 2-5A binding site and entire nine ankyrin

68 repeats and produces a peptide with only 2-5A binding activity, but no enzymatic activity

(Fig. 3.1).

Figure 3.1 Expression of dominant negative RNase L in L929 cells

The PD12-pcDNAneo and empty vectors were transfected into L929 cells by using the lipofectin reagent (Life Technologies), followed by selection in the medium containing G418. 2-5A binding assay (150 μg/sample) was used to determine the expression of PD12. Clones of No. 5-1 and 6-3 were used in this project.

70 3.2.1.2 Cell culture and insulin treatment

L929 PD12 cells were cultured in RPMI 1640 medium (Cleveland Clinic,

Cleveland, Ohio) supplemented with 10% (V/V) fetal bovine serum (FBS, Atlanta

Biologicals, Inc., Flowery Branch, Georgia), 100U/ml penicillin-streptomycin.

Cells were starved in serum free media for 24 hours prior to insulin stimulation.

Insulin was diluted from stock solutions in PBS without additives and added to cells at a final concentration of 100 nM for 10, 30, 60 mins at 37°C. Unstimulated cells were served as controls. After treatment, the cells were rinsed and harvested with ice-cold Tris-buffered saline (TBS) on ice. After centrifuging, the cell pellets were frozen on dry ice and stored in -80 °C for Western blot analysis.

3.2.2 MG132 treatment of MEFs

MG132 (carbobenzoxyl-L-leucyl-L-leucyl-L-leucinal) is a triterpene proteasome inhibitor. It is a peptide aldehyde that inhibits 20S proteasome activity by covalently binding to the active site of the beta sub-units and effectively blocks the proteolytic activity of the 26S proteasome complex (Figure 3.2) and reduces the degradation of ubiquitin- conjugated proteins in mammalian cells.

To determine the effect of RNase L on ubiquitination of proteins in cells, primary

RNase L+/+ and -/- MEFs were treated in the absence or presence of MG132 and analyzed the profile of ubiquitinated proteins by Western blot analysis.

In these experiments, primary MEFs were cultured as described in 2.2.6. Before

MG132 treatment, MEFs were starved in serum free media for 24 hours. MG132 in DMSO was added to the cells at a final concentration of 10 μM for 2 hours. Cells treated with

71 DMSO alone served as controls. After the treatment, cells were rinsed with ice-cold Tris- buffered saline (TBS) on ice and then were harvested. The cell pellets were frozen on dry ice and stored in -80°C for Western blot analysis.

Figure 3.2 The inhibitory role of MG132

(Guo N, Peng Z. Asia Pac J Clin Oncol. 2013)

73 3.2.3 Western blot analysis

Western blot analysis was performed as described in 2.2.8.2 and the antibodies used in the experiments are listed in Table 3.1.

Antibody Isotype Catalog NO. Company

β-Actin Mouse sc-47778 Santa Cruz

β Tubulin Mouse sc-5274 Santa Cruz

Insulin Receptor-α Mouse sc-710 Santa Cruz

Insulin Receptor-β Mouse sc-57342 Santa Cruz

mTOR Rabbit #2983 Cell Signaling

p-Akt (Thr308) Rabbit #4056 Cell Signaling

p-IRβ (Tyr1150) Mouse sc-81500 Santa Cruz

p-IRβ (Tyr1162) Rabbit sc-25103 Santa Cruz

p-IRS-1 (Ser302) Rabbit #2384 Cell Signaling

p-IRS-1 (Tyr895) Rabbit #3070 Cell Signaling

p-VCP Rabbit SAB4504738 Sigma

Ubiquitin Mouse sc-8017 Santa Cruz

VCP Mouse sc-20799 Santa Cruz

Table 3.1 Antibodies used in the project

74 3.3 Results

3.3.1 Overexpression of the 2-5A binding domain affects the insulin signaling pathway

To determine if the RNase enzymatic function of RNase L affects the insulin signaling pathway, we examined the effect of 2-5A binding domain, a dominant negative mutant, on activation of IR, IRS-1 and Akt, the major components of insulin signaling

(Figure 3.4). As shown in Figure 3.3, the overexpression of 2-5A binding domain up regulated the level of IR-β and enhanced its phosphorylation, suggesting that 2-5A binding domain may be involved in the expression of IR. However, the phosphorylation of IRS-1 at Tyr 895 and Ser 302 was down regulated in PD12 cells. Nevertheless, the activation of

Akt in PD12 cells was slightly enhanced compared to that in wild type cells. Interestingly, the total Akt protein was found to be down regulated in the cells, implicating that the 2-5A binding domain of RNase L may directly affect the expression of Akt, but the molecular mechanism needs to be further investigated. As described above, the 2-5A binding domain contains 9 Ankyrin-repeats that are typical protein-protein interaction domains. Thus, this observation suggests that RNase L may regulate the expression of other proteins through the interaction of its Ankyrin-repeats to the factors in the transcriptional and translational systems. Further investigation of the subjects is warranted.

Figure 3.3 Overexpression of the 2-5A binding domain affects the expression of IR

PD12 and wild type L929 cell extracts were subjected to Western blot analysis with antibodies for the IR Precursor and IR-β (A) (Santa Cruz). Quantitative analyses were performed by using NIH image software for the ratios of IR Precursor /β-Tubulin (B) and IR-β/β-Tubulin (C).

Figure 3.4 Overexpression of the 2-5A binding domain affects the insulin signaling

PD12 and wild type L929 cells were treated with 200 nM insulin (Sigma) for various times. The cell extracts were subjected to Western blot analysis with antibodies for p-IR β, p-IRS- 1(Santa Cruz), p-IRS-1 and p-AKT (A) (Cell Signaling). Quantitative analyses were

77 performed by using NIH image software for the ratios of p-IR β (Tyr 1150)/mTOR (B), p- IR β (Tyr 1162)/mTOR (C), p-IRS-1 (Ser 302)/ mTOR (D), p-IRS-1 (Tyr 895)/ mTOR (E) and p-Akt (Thr 308)/ mTOR (F).

78 3.3.2 RNase L impacts phosphorylation of P97/VCP and ubiquitination in MEFs

Interestingly, the differential levels of phosphorylated P97/VCP were also observed in RNase L +/+ and -/- MEFs (Fig. 3.5A). Because of its role in ERAD, we next determined the effect of RNase L on ubiquitination of proteins in primary RNase L+/+ and -/- MEFs treated with or without 2 μM MG132, a commonly used proteasome inhibitor, and analyzed the profile of ubiquitinated proteins by Western blot analysis. As shown in Figure 3.5C, the levels of ubiquitinated proteins in the cytoplasm of RNase L+/+ and -/- MEFs were significantly different, implicating the involvement of RNase L ubiquitin-proteasome system.

Figure 3.5 RNase L mediates phosphorylation of P97/VCP and ubiquitination in

MEFs

The phosphorylation status of VCP in RNase L +/+ and -/- primary MEFs was examined by Western blot analysis with an antibody for p-VCP (A) (Sigma). Both types of MEFs were treated with 2μM of MG132 (Cayman) for 2 hours. The ubiquitinated protein profiles were examined in the cell extracts by Western blot analysis with an antibody for Ubiquitin (Santa Cruz). Quantitative analyses were performed by using NIH image software for the ratios of p-VCP/VCP(B) and Ub proteins (D) in different cell types.

80 3.3.3 Inhibiting the proteasome enhanced the activation of IRS-1 in RNase L deficient

MEFs

To further demonstrate the correlation of RNase L with the ubiquitin-proteasome system, starved RNase L +/+ and -/- MEFs were pre-treated with 2 μM MG132 for 2 hours, followed by insulin stimulation. As expected, the phosphorylation of IRS-1 at Ser 302 was significantly increased after the proteasome system was inhibited (Fig. 3.6), suggesting that

RNase L may impact insulin response by regulating the ubiquitin-proteasome pathway via effecting the phosphorylation of VCP.

Figure 3.6 Inhibition of the proteasome system enhanced the activation of IRS-1

RNase L +/+ and -/- primary MEFs were treated with 2μM of MG132 (Cayman) for 2 hours, followed by 200 nM insulin (Sigma) for 40 min. The cell extracts were subjected to Western blot analysis with an antibody for p-IRS-1 (Ser 302) (Cell Signaling). Quantitative analyses were performed by using NIH image software for the ratios of p-IRS-1 (Ser 302)/ β-Tubulin (B).

82 3.4 Discussion

The data in Chapter II showed that the lack of RNase L caused the down regulated level of IR-β, implicating that RNase L may impact on the cleavage of IR. To explore how

RNase L affects the cleavage of the IR precursor to produce α- and β-subunits, we examined the level and activation of IR-β in PD12 cells. Interestingly, we observed that overexpressing of the 2-5A binding domain enhanced the phosphorylation of IR by upregulating the expression of IR-β. The N-terminal half of RNase L is a highly conserved protein/protein interaction domain containing nine Ankyrin repeats and the 2-5A binding domain is located at the 2 to 4 Ankyrin repeats [151]. So far, the only well-established function of the domain is for activating RNase L after binding 2-5A. Although how RNase

L regulating the level of IR-β in the cells after insulin stimulation remains vague, it is possible that RNase L may contribute to the differential changing of the IR-β protein level through the protein-protein interaction to manipulate the production of other proteins based on the data obtained from PD12 cells.

RNase L has a conserved protein kinase homology at the C-terminal half, suggesting that RNase L may have specific kinase activity, although it has not been demonstrated yet. The N-terminal domain functions as a repressor of the RNase domain in the C terminal half (Figure 1.3) and the binding of 2-5A to the ARD induces a conformational change of RNase L, causing the N-terminal repressor domain to release from the PK and RNase domain, leading to the activation of RNase L. The mutant expressed in PD12 cells contains the entire nine Ankyrin repeats with the 2-5A binding site and produces a peptide with only 2-5A binding activity. The mutant peptide is able to compete with the full length of RNase L for binding with 2-5A, resulting in decreasing

83 activation of RNase L. Interestingly, we observed that the phosphorylation of IRS-1 and

Akt, the key enzymes in the insulin signaling pathway, was still relatively low upon insulin stimulation although the activation of IR-β was enhanced. These results suggest that RNase

L may not only mediate the cleavage of the IR precursor, but also function as an enzyme to impact the insulin signaling.

The 2-5A is normally synthesized by 2-5A synthetase (OAS) which can be activated by binding a dsRNA generated from virus [9]. In our study, we use insulin to stimulate the cells other than viral infection. Therefore, the 2-5A should be needed to activate RNase L in the absent of dsRNA. The overexpression of 2-5A binding domain impaired insulin sensitivity, which implicates that certain molecules need to bind the 2-5A binding domain of RNase L for its kinase activity. It has been reported that Theiler's murine encephalomyelitis virus (TMEV) L*(L-Star) protein, an 18kDa protein encoded by an alternative open reading frame overlapping the L-, VP4- and VP2-coding regions of the

ORF encoding the viral polyprotein [164], can inhibit RNase L through direct protein- protein interaction [165]. The binding of L* to the Ankyrin repeats 1 and 2 of RNase L inhibits 2-5A binding to RNase L [166]. In our results, it is possible that RNase L can mediate the activation of the insulin signaling pathway through the protein-protein interaction of ARD domain.

It has been well established that activation of IR is responsible for the Tyr- phosphorylation of IRS-1, which subsequently activates the proteasome pathway, leading to IRS-1 degradation [171]. Intriguingly, our results revealed that although IR was not activated, the Ser-phosphorylated IRS-1 was still degraded in the cell deficient RNase L, suggesting that the degradation of IRS-1 may be caused by the inactivation of IR rather

84 than activation of IR. Apparently, RNase L could also be a regulator of the IRS-1 degradation and lack of RNase L may promote the ubiquitin-proteasome system to degrade

IRS-1.

CHAPTER IV

SUMMARY AND FUTURE DIRECTION

4.1 Summary of the project

In this project, we found that RNase L might attenuate the insulin signaling pathway through regulating the cleavage of the IR precursor. Deficiency of RNase L in cells may cause the altered cleavage of the IR precursor to produce subunit-α and -β. The activation of these downstream components in the insulin signaling pathway may be mediated by

RNase L. Interestingly, we observed that RNase L not only impacts IR, but also affects the phosphorylation of IRS-1 under certain conditions, suggested that RNase L may function through alternative pathways to promote the phosphorylation of IRS-1, although this hypothesis needs to be further investigated. Our results obtained from this project provides a better understanding of RNase L functions, and open a new horizon for its role in metabolic studies besides in the virology field. Furthermore, the effect of RNase L on the ubiquitin-proteasome system confers this enzyme as a specific target for studying protein homeostasis and a variety of human diseases.

86 4.2. Future direction

Up to date, the well-established role of RNase L is to function as a key enzyme in the 2-5A system of IFN against viral infection and cell proliferation. Our results provide a new insight into the role of RNase L in the cell metabolism. Although we have found that

RNase L mediates the insulin signaling pathway, the contribution of RNase L remains to be fully understood. How is RNase L involved in the cleavage process of the IR precursor?

If it is through mediating the ubiquitin/proteasome system, does RNase L affect the ubiquitination or proteasome activity? It may be neither of the two ways because RNase L deficient cells have a significantly higher level of phosphorylated VCP that promotes ubiquitated proteins moving from the membrane of ER to cytoplasm where they are degraded. Therefore, further investigation of the role of RNase L in the ubiquitin/proteasome system is also an interesting research project. Below are some thoughts about elucidation of RNase L functions in the metabolic mechanism of glucose and lipid.

4.2.1 Determine the role of RNase L in insulin sensitivity in the adipose tissue and cells

Adipose tissue is one of the tissues responses to the insulin and uptake the glucose through the GLUT4 dependent pathway [134]. 3T3-L1 is a mouse embryonic fibroblast cell line, which can differentiate to adipocyte-like cells. RNase L knockout 3T3-L1 cells can be generated by using the CRISPR/Cas9 technology. RNase L deficient 3T3-L1 cells will be induced to differentiate lipid cells. Both RNase L +/+ and -/- adipocytes derived from

3T3 L-1 will be used to determine the role of RNase L in insulin sensitivity. Primary

87 adipocytes isolated from RNase +/+ and -/- mice can also be used to further confirm the results.

4.2.2 Investigate the effects of RNase L on insulin signaling in vivo

The information obtained from in vitro studies needs to be demonstrated by an in vivo model. Thus, we will use RNase L +/+ and -/- mice to confirm our results from the cells.

RNase L +/+ and -/- mice can be injected with insulin. After certain time, the activation of the insulin signaling pathway in the tissues such as the liver, adipose and others can be examined as described above. Furthermore, the effect of RNase L on insulin sensitivity and the blood glucose level can be determined. We could also create a type I diabetes model in both types of the mice and then examine the effects of RNase L on insulin sensitivity.

4.2.3 Test the kinase activity of RNase L

The structure of RNase L indicates that there are nine Ankyrin repeats associated with the 2-5A binding, an RNase domain to degrade target RNAs and a protein kinase-like domain, of which the function has not been demonstrated yet to date. Interestingly, our results suggest that RNase L may have kinase activity because the profiles of phosphorylated proteins in the RNase L +/+ and -/- cells and tissues are remarkably different, suggesting the possibility that RNase L may have a kinase activity or indirectly impact the function of other kinases. Therefore, determination of RNase L kinase activity is one of the next research goals in this project.

88 4.2.4 Examine the RNase L level in the samples of patients with metabolic disorders

Our ultimate goal is to determine the value of RNase L in the clinic because it is a target that mediates the insulin signaling pathway. We assume that RNase L is essential for the insulin sensitivity and glucose uptake, RNase L deficiency may cause the insulin resistance, leading to Type II diabetes. The first thing we need to know is the correlation of the level of RNase L and metabolic syndromes in patients. RNase L levels in the blood samples from patients with obesity, insulin resistance or Type II diabetes will be examined and compared with that in health population. Based on our current results, the level of

RNase L would be lower in patients with diabetes than that in health people. If that is the case, RNase L may be considered as a promising biomarker for the insulin resistance and diabetes. Novel therapeutic strategies for metabolic disorders could be developed by regulating the level and activity of RNase L.

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