Chun Zhou

Serge Przedborski

Intrabody and Parkinson's disease

The intrabody technology has become a promising therapeutic avenue for a variety of incurable diseases. This technology is an intracellular application of gene-engineered antibodies, aimed at ablating the abnormal function of intracellular molecules. Parkinson's disease (PD) is a common neurodegenerative disease with no cure. Recent studies have explored possible intrabody applications against alpha-synuclein (α-syn), whose misfolding is believed to cause a familiar form of PD. Here, we review the origin, production, and therapeutic mechanisms of intrabodies and the potential of intrabody protection against α-syn toxicity. Furthermore, we propose possible intrabody applications against leucine-rich repeat kinase 2 (LRRK2), whose mutations are the most frequent known cause of familial and sporadic PD.

Introduction

Recently, the intrabody technology has emerged as a novel tool to modulate the function of intracellular proteins [START_REF] Lobato | Intracellular antibodies as specific reagents for functional ablation: future therapeutic molecules[END_REF]. Moreover, this technology has been applied in various research settings that model complex human pathological conditions such as Parkinson's disease (PD), the second most common neurodegenerative disorder after Alzheimer's dementia [START_REF] Dauer | Parkinson's disease: mechanisms and models[END_REF]. The pathological features of PD include a loss of dopaminergic neurons in the substantia nigra pars compacta [START_REF] Braak | Nigral and extranigral pathology in Parkinson's disease[END_REF] and the formation of the Lewy body (LB), a cytoplasmic proteinaceous inclusion composed principally of alpha-synuclein (α-syn) [START_REF] Spillantini | α-Synuclein in Lewy bodies[END_REF]. Currently, while medicines such as Levodopa may help to manage symptoms, to date, no treatment has been shown to slow or stop the progression of PD.

Although PD arises essentially as a sporadic condition, it is occasionally inherited [START_REF] Vila | Genetic clues to the pathogenesis of Parkinson's disease[END_REF]. In the past decade, studies on various proteins whose genetic mutations cause familial PD, such as α-syn, parkin, DJ-1, PTEN-induced putative kinase 1 (PINK1), and leucine-rich repeat kinase 2 (LRRK2), have largely improved our understanding of possible pathogenic mechanisms of PD and have provided us with potential therapeutic targets for PD.

Based on a paucity of available intrabody studies in PD, the intrabody therapy for this common disorder has unambiguously a long path to explore. However, it holds one of the promises of a cure for PD since it aims to remedy the pathogenic abnormalities at the molecular level. In this review, to provide a general background, we will first discuss the origin, production and therapeutic mechanisms of intrabody. Then, we will discuss intrabody studies in PD, with focus on α-syn. Finally, we will propose potential intrabody applications to prevent the cellular toxicities caused by mutant LRRK2, shedding light on intrabody studies in new PD-related scenario.

Antibody and scFv

Antibodies, or immunoglobulins, are one of the major protein effectors of an immune system. A typical antibody

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ACCEPTED MANUSCRIPT 4 molecule consists of two heavy chains and two light chains, connected by disulfide bonds. Compared to the constant regions (C H and C L ), which share high homology among different antibodies, the variable regions (V H and V L ) are extremely variable, forming a unique three-dimensional structure, the antigen-binding site or Fv (Fig. 1). The Fv binds a structurally complementary molecule, known as an antigen (Ag). The high variability of Fv makes it possible for a natural antibody pool to recognize a broad range of antigens.

Antibodies have been broadly used for disease diagnosis and therapy mainly due to their high specificity and high affinity. An antigen determinant, also termed epitope, is the portion of an antigen that binds to an antibody.

Antibody specificity is the property that enables antibodies to discriminate between two different antigen determinants. The strength with which an antibody binds to an antigen determinant is called affinity, usually described by the dissociation constant, K D (mol/L, M). Since Kohler & Milstein described the monoclonal antibody (MAb) technology in 1975 [START_REF] Kohler | Continuous cultures of fused cells secreting antibody of predefined specificity[END_REF], an ideal MAb with both high specificity and high affinity has been believed to be a "magic bullet" for target therapy. Although initial clinical trials with MAbs showed some promising antitumor effects [START_REF] Miller | Treatment of B-cell lymphoma with monoclonal anti-idiotype antibody[END_REF], MAb therapy is hindered by the following major obstacles: human antimouse antibody (HAMA) responses generated by the immunogenicity of mouse MAbs, difficulties in identifying specific targets such as tumor-specific antigens, and the low efficacy of antibody enrichment at affected tissues [START_REF] Von | Monoclonal antibody therapy for cancer[END_REF].

To overcome the above issues, in vitro antibody display technologies have been developed to display a large repertoire of gene-engineered antibodies (>10 9 members) on the surfaces of phage, yeast, or others, for rapid screening of a specific antibody of interest [START_REF] Griffiths | Isolation of high affinity human antibodies directly from large synthetic repertoires[END_REF][START_REF] Winter | Making antibodies by phage display technology[END_REF][START_REF] Feldhaus | Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library[END_REF][START_REF] Hanes | In vitro selection and evolution of functional proteins by using ribosome display[END_REF][START_REF] Daugherty | Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface[END_REF][START_REF] Chen | Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS)[END_REF]. Gene-engineered antibodies refer to recombinant antibody molecules that are generated by expressing the cDNA of engineered antibody fragments, such as "V H -linker-V L " (Fig. 1). Usually, the engineered antibody fragments contain at least one complete or partial antigen binding site to retain antigen-binding function. Gene-engineered antibodies consist of many types of antibody molecules,

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such as single-chain Fv (scFv, ~25 kDa) and Fabs (antigen binding fragments, ~55 kDa) (Fig. 1). Here, throughout this review, for the simplicity of demonstration, we use scFv to represent all other gene engineered antibodies.

ScFv, a key molecule of gene engineered antibodies, is composed of the V H and V L regions, which are connected by a flexible short linker with a major form of (Gly 4 Ser) 3 (Fig. 1) [START_REF] Hudson | Recombinant antibodies for cancer diagnosis and therapy, Expert[END_REF]. ScFv is attractive due to the following advantages. First, it has a small size of ~25 kDa (vs ~150 kDa of a typical antibody), yielding rapid renal elimination and better tumor tissue penetration [START_REF] Begent | Clinical evidence of efficient tumor targeting based on single-chain Fv antibody selected from a combinatorial library[END_REF][START_REF] Yazaki | Biodistribution and tumor imaging of an anti-CEA single-chain antibody-albumin fusion protein[END_REF][START_REF] Olafsen | Optimizing radiolabeled engineered anti-p185HER2 antibody fragments for in vivo imaging[END_REF]. Yet, it retains good antigen-binding specificity because the antigen-binding site is unaltered. Second, to generate a scFv, the coding gene segments of V H and V L regions are isolated either from a hybridoma expressing the MAb with desired specificity [START_REF] Huston | Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli[END_REF], or from a large antibody library with more than 10 9 members of diverse scFvs; the latter gives the potential for screening scFvs against virtually any possible antigen in a short time [START_REF] Marks | By-passing immunization. Human antibodies from V-gene libraries displayed on phage[END_REF]. Third, most antibody libraries contain scFvs as human proteins, which do not induce HAMA responses, because these scFvs are made from the coding genes of V H and V L regions from human lymphocytes. Fourth, optimized scFv functional characteristics, such as high affinity, long retention time (the capability to stay in antigen-binding status), and enhanced protein stability, can be achieved by further mutagenesis of the scFv cDNAs and by further selection from sub-libraries containing these mutated scFvs [START_REF] Adams | Generating improved single-chain Fv molecules for tumor targeting[END_REF]. Clearly, scFvs and other gene-engineered antibodies represent a type of promising reagent for targeted therapy. Furthermore, innovative technologies have largely improved the likelihood of an antibody being a "magic bullet" and are still undergoing further development, especially in the improvement of minimizing immunogenicity, in increasing specificity, affinity, and stability, and in employing new application scenarios. For example, recently, the application of scFv inside of human cells has become a completely novel type of molecular therapy, intrabody therapy.

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Intrabody

Intrabodies, or intracellular antibodies, here refer to gene-engineered antibodies (mainly scFvs) that are expressed intracellularly to modulate the function of their intracellular targets. Usually, cell transfections or infections with scFv cDNA-containing plasmids or viruses are the means to express scFvs intracellularly. Clearly, the intrabody technology is an intracellular application of gene-engineered antibodies.

Challenges

To use scFvs intracellularly, a major obstacle is the fact that the cytoplasmic reducing environment prevents the formation of disulfide bonds. As shown in Fig. 1, disulfide bonds are necessary for optimized folding of the antigen binding site of scFv [START_REF] Wirtz | Intrabody construction and expression III: engineering hyperstable V(H) domains[END_REF]. Thus, for most scFvs isolated from antibody libraries or from hybridomas, the antigen binding capacities are more or less decreased or even completely lost when these scFvs are expressed as intrabodies in the cytosol (Fig. 1) [START_REF] Ramm | Removal of the conserved disulfide bridges from the scFv fragment of an antibody: effects on folding kinetics and aggregation[END_REF][START_REF] Worn | Stability engineering of antibody single-chain Fv fragments[END_REF]. For this reason, to select the scFvs that can bind their antigens inside cells, researchers have developed the following two strategies: a) in vitro screening of scFvs from antibody libraries followed by further intracellular validation in mammalian cells [START_REF] Zhou | A human single-chain Fv intrabody blocks aberrant cellular effects of overexpressed alpha-synuclein[END_REF][START_REF] Lynch | An scFv intrabody against the nonamyloid component of alpha-synuclein reduces intracellular aggregation and toxicity[END_REF]; b) intracellular scFv screening to identify intracellular functional scFvs directly [START_REF] Visintin | Selection of antibodies for intracellular function using a two-hybrid in vivo system[END_REF][START_REF] Portner-Taliana | In vivo selection of single-chain antibodies using a yeast two-hybrid system[END_REF][START_REF] De | Analysis of the interaction between single-chain variable fragments and their antigen in a reducing intracellular environment using the two-hybrid system[END_REF]. As a result, the most critical parameter of a successful intrabody is its intrinsic folding capacity under a reducing environment, rather than its in vitro affinity [START_REF] Worn | Correlation between in vitro stability and in vivo performance of anti-GCN4 intrabodies as cytoplasmic inhibitors[END_REF]. In addition, there are other important challenges for intrabody therapy, such as the efficacy of gene delivery, which are not in the scope of this review.

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Production of intrabodies

As mentioned above, intrabodies can be generated by using direct intracellular scFv screening methods [START_REF] Visintin | Selection of antibodies for intracellular function using a two-hybrid in vivo system[END_REF][START_REF] Portner-Taliana | In vivo selection of single-chain antibodies using a yeast two-hybrid system[END_REF][START_REF] De | Analysis of the interaction between single-chain variable fragments and their antigen in a reducing intracellular environment using the two-hybrid system[END_REF]. However, while these methods are waiting to be widely applied and tested, the following strategy remains as a feasible and widely used method to generate functional intrabodies (Fig. 2).

First, antigens are usually produced as recombinant proteins by expressing the antigen cDNAs in bacteria, followed by protein purification. Of note, some eukaryotic proteins require post-translational modifications for maximal folding/activity, whereas bacteria do not have the machinery required to accomplish complex modifications. Therefore, when these modifications are necessary, eukaryotic cells, such as yeast cells and insect cells, can be used for the production of recombinant proteins. In the cases that recombinant proteins are not readily available, or scFvs against specific epitopes are needed, synthesized peptides are often used as antigens. Numerous antibody libraries are available for scientific studies, such as synthetic phage libraries (the Tomlinson I and J antibody libraries, MRC Center for Protein Engineering, Cambridge, England) and naïve yeast libraries [START_REF] Feldhaus | Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library[END_REF]. With the antigen and antibody libraries available, one can screen a set of scFvs specifically against an antigen according to standard protocols [START_REF] Griffiths | Isolation of high affinity human antibodies directly from large synthetic repertoires[END_REF][START_REF] Feldhaus | Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library[END_REF]. Once a set of binders are selected, it is important to characterize their in vitro features, including specificity, affinity, and epitopes. From this in vitro characterization, a set of scFvs with good antibody features usually can be acquired. However, only the scFvs with functionally intracellular binding to their specific targets can be used as intrabodies (see later discussion in the section of in vitro versus in situ characterization of scFvs).

Therapeutic strategies

Since 1988, when a study showed that intracellular expression of the cDNAs of antibody heavy and light

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chains against yeast alcohol dehydrogenase I successfully inactivated the enzyme in vivo [START_REF] Carlson | A new means of inducibly inactivating a cellular protein[END_REF], the intrabody technology has been broadly employed in the studies involving human diseases, mainly AIDS and cancer. The following strategies are used for intrabodies to ablate or restore the function of intracellular molecules. First, intrabodies can block the interaction between their target proteins and other molecules, such as proteins and nucleic acids. For example, human immunodeficiency virus type1 (HIV-1) protein Tat functions through binding with human cyclinT1 to promote the transcription elongation of HIV-1. By disrupting the molecular interaction between HIV-1 Tat and human cyclinT1, anti-human cyclinT1 intrabodies effectively block HIV-1 replication without causing cellular toxicity [START_REF] Bai | Inhibition of Tat-mediated transactivation and HIV-1 replication by human anti-hCyclinT1 intrabodies[END_REF]. Second, by retaining their targets in a particular subcellular compartment, such as endoplasmic reticulum (ER), intrabodies can prevent their target molecules from being at their physiological locations to function [START_REF] Cattaneo | The selection of intracellular antibodies[END_REF]. In fact, by attaching an organelle-targeting sequence to an intrabody, the expression of intrabodies at specific subcelluar compartments can be achieved, such as in nuclei, ER and lysosomes [START_REF] Biocca | Intracellular immunization: antibody targeting to subcellular compartments[END_REF]. For instance, tagged with the ER-retention signal (SEKDEL), an anti-gp120 intrabody has been used to trap gp120, an envelope protein of HIV-1, inside the lumen of ER to prevent the assembly of new infectious virions [START_REF] Marasco | Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody[END_REF]. Third, intrabodies can restore normal protein function that has been lost due to mutagenesis. For instance, p53 is a tumor suppressor gene that is mutated in ~ 50% of cancers. Mutation of p53 results in a loss of function of p53 protein by diminishing its DNA binding activity, which is required for its tumor suppressor function. An anti-p53 intrabody has been shown to restore the normal conformation and DNA binding activity of p53 and thus to suppress tumor progression [START_REF] Caron De | Restoration of transcriptional activity of p53 mutants in human tumour cells by intracellular expression of anti-p53 single chain Fv fragments[END_REF]. Fourth, intrabody can prevent aberrant protein conformation and protein aggregation. This aspect will be discussed later in the section related to α-syn.

A C C E P T E D M A N U S C R I P T Advantages

Obviously, intrabodies are valuable for both functional studies of a given intracellular molecule and the development of novel therapeutics. In fact, compared to other similar technologies aimed at blocking protein function, such as RNA interference (RNAi), intrabody technology has the advantage of modifying certain domains of a protein without affecting other domains because intrabodies have domain-specific binding capabilities. In addition, intrabodies can also modify the three-dimensional structure of a protein and thus are important for both stabilizing active protein conformations and correcting aberrant protein conformations. The abnormal protein conformations are closely related to the pathogenic mechanisms of neurodegenerative diseases, such as PD. Moreover, for proteins with relatively long half-lives or for proteins whose mRNAs have short half-lives, RNAi may be less effective while intrabodies can still function effectively through direct binding to the proteins.

Efficacy in animal studies and clinical trails

Geared toward a molecular therapy for human diseases, the intrabody technology has been applied in animal studies and clinical trials. For instance, the neutralizing anti-Ras intrabody Y13-259, which induces in vitro transient phenotypic reversion of rodent fibroblasts transformed by ras, an oncogene, has been shown to invoke sustained tumor regression in nude mice [START_REF] Cochet | Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression[END_REF]. Moreover, an ER-targeted intrabody against Tie-2, a receptor that is up-regulated in capillaries during neovascularization processes produced in tumors, has been shown to significantly inhibit tumor growth in mouse models [START_REF] Popkov | Targeting tumor angiogenesis with adenovirus-delivered anti-Tie-2 intrabody[END_REF]. More recently, the intrabody EM48 has been shown to suppress neuropil aggregates in a mouse model of Huntington's disease (HD) [START_REF] Wang | Suppression of neuropil aggregates and neurological symptoms by an intracellular antibody implicates the cytoplasmic toxicity of mutant huntingtin[END_REF], thus indicating promising applications of intrabodies in neurodegenerative diseases. In the Phase I clinical trial using an ER-targeted

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anti-erbB-2 intrabody, which had been previously shown to prolong survival in human ovarian cancer xenograft models [START_REF] Deshane | Targeted tumor killing via an intracellular antibody against erbB-2[END_REF], of 13 ovarian cancer patients with erbB-2 overexpression, 5 (38%) had stable disease and 8 (61%) had evidence of progressive disease [START_REF] Alvarez | A cancer gene therapy approach utilizing an anti-erbB-2 single-chain antibody-encoding adenovirus (AD21): a phase I trial[END_REF]. Except for showing that the intrabody approach is feasible for human application with very limited side effects, this clinical study indicates that compared to cellular and animal studies, the intrabody technology, including gene delivery methods, needs to be further developed to achieve a dramatic effect for the therapy of human diseases.

Intrabody and PD

In light of the initial successful application of intrabodies in the studies of AIDS and cancer, the intrabody technology has been employed in the studies of neurodegenerative diseases with the hopes of preventing abnormal protein aggregation. For instance, in HD, the polyglutamine expansion in the huntingtin (htt) protein causes htt aggregation, which is thought to be toxic to cells. It has been shown that anti-htt intrabodies can inhibit mutant htt aggregation and thus rescue cells from death [START_REF] Lecerf | Human single-chain Fv intrabodies counteract in situ huntingtin aggregation in cellular models of Huntington's disease[END_REF][START_REF] Colby | Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody[END_REF] and the anti-htt intrabody C4 shows in vivo efficacy of protection in a Drosophila model of HD [START_REF] Wolfgang | Suppression of Huntington's disease pathology in Drosophila by human single-chain Fv antibodies[END_REF]. Moreover, the intrabody EM48 has been shown to suppress neuropil aggregates in a HD mouse model [START_REF] Wang | Suppression of neuropil aggregates and neurological symptoms by an intracellular antibody implicates the cytoplasmic toxicity of mutant huntingtin[END_REF]. Another example in the study of Alzheimer's disease is the use of intrabodies to inhibit the generation of the toxic amyloid beta-peptide, by blocking the beta-secretase cleavage site or by retaining the beta-amyloid precursor protein in ER [START_REF] Paganetti | beta-site specific intrabodies to decrease and prevent generation of Alzheimer's Abeta peptide[END_REF].

In the following sections, we will discuss the intrabody application in PD. Although the intrabody technology can be used for studying all PD-related proteins, here, we focus on α-syn and LRRK2, two proteins that cause autosomal dominant PD, to demonstrate a proof of principle of existing and future intrabody applications in PD.

A C C E P T E D M A N U S C R I P T α-Syn

α-Syn is a 140 amino acid protein that belongs to the synuclein protein family, which also includes β-syn and γ-syn. Previous studies using both optical methods and NMR have shown that α-syn exhibits a random-coil structure (or natively unfolded structure) under physiological conditions [START_REF] Weinreb | NACP, a protein implicated in Alzheimer's disease and learning, is natively unfolded[END_REF][START_REF] Eliezer | Conformational properties of alpha-synuclein in its free and lipid-associated states[END_REF]. Mutations in α-syn cause a rare form of PD. The fact that α-syn is the major component of LB, a hallmark of PD, has put α-syn under intense study that eventually led to the consensus that protein aggregation is one of the major pathogenic mechanisms of PD. Although it is debatable whether LBs are toxic or protective and which forms of aggregated α-syn such as oligomers versus fibrils mediate toxicity, it is clear that α-syn is a natively unfolded protein that may acquire a gain of toxic function due to self-aggregation [START_REF] El-Agnaf | Aggregates from mutant and wild-type alpha-synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of beta-sheet and amyloid-like filaments[END_REF][START_REF] Conway | Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease[END_REF][START_REF] Giasson | Mutant and wild type human alpha-synucleins assemble into elongated filaments with distinct morphologies in vitro[END_REF][START_REF] Spillantini | α-synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with Lewy bodies[END_REF]. Therefore, the development of anti-α-syn intrabodies may be potentially useful in blocking α-syn aggregation and therefore protect cells from toxic α-syn aggregates.

Natively unfolded proteins have been categorized as a novel protein family -naturally unfolded (or intrinsically unstructured) protein family. It is hypothesized that these natively unfolded proteins often acquire ordered three-dimensional structures upon binding to their partners, such as proteins and nucleic acids, and modify the function of their partner molecules [START_REF] Dyson | Intrinsically unstructured proteins and their functions[END_REF][START_REF] Uversky | Natively unfolded proteins: a point where biology waits for physics[END_REF]. Based on the available knowledge of natively unfolded proteins and previous studies on α-syn, we propose a simplified paradigm of possible α-syn structure-function relations as a working hypothesis for developing potential protective intrabodies against PD (Fig. 3). In this schematic, α-syn exists physiologically as a natively unfolded protein with the potential of transforming into the following three ordered structures. The available experimental evidence shows that the N-terminal sequence of α-syn binds to acidic lipids and forms an α-helical structure. One hypothesis is that α-syn functions on cellular membranes once it acquires an ordered structure through binding to the membranes. In addition, like other natively unfolded proteins, α-syn may also bind to other multiple targets to play an important role as a modifier of Based on the above model, it is tempting to develop intrabodies that can specifically block the self-binding of α-syn without affecting the binding of α-syn to its physiological partner molecules (Fig. 3). In the pioneer studies aimed at this goal, various scFvs against α-syn have been generated and we have listed the majority of these in Table 1. In the following, we will discuss several critical issues based on the available studies of anti-α-syn scFvs and intrabodies to shed light on future studies.

Source of intrabodies: As potential therapeutics for human diseases, intrabodies are often generated by screening scFvs from human antibody libraries rather than by cloning scFv gene segments from mouse hybridomas. Current available anti-α-syn scFvs/intrabodies are selected from three phage libraries and one yeast library (Table 1). ScFvs F8 [START_REF] Emadi | Inhibiting aggregation of alpha-synuclein with human single chain antibody fragments[END_REF], D10 [START_REF] Zhou | A human single-chain Fv intrabody blocks aberrant cellular effects of overexpressed alpha-synuclein[END_REF], 6E [START_REF] Barkhordarian | Isolating recombinant antibodies against specific protein morphologies using atomic force microscopy and phage display technologies[END_REF], D5 [START_REF] Emadi | Isolation of a human single chain antibody fragment against oligomeric alpha-synuclein that inhibits aggregation and prevents alpha-synuclein-induced toxicity[END_REF] are selected from two synthetic libraries that contain synthesized variable regions of scFvs. On the other hand, scFvs NAC3, NAC24, and NAC32 [START_REF] Lynch | An scFv intrabody against the nonamyloid component of alpha-synuclein reduces intracellular aggregation and toxicity[END_REF] are selected from a yeast library that is composed of scFv gene segments from the mRNA pool of 58 human adult spleens and lymph nodes. In addition, scFvs 3, 14 and 15 [START_REF] Maguire-Zeiss | Identification of human alpha-synuclein specific single chain antibodies[END_REF] are from a phage library generated using the mRNAs from human peripheral blood lymphocytes of a large (>100) pool of donors. Thus, all the scFv libraries currently used for generating anti-α-syn scFvs are naïve libraries in which scFv gene segments are from unimmunized individuals or are artificially synthesized. This suggests that, despite the unique unfolded structure of α-syn, it is feasible to screen anti-α-syn scFvs from current available large scFv libraries, no matter what display platform a library adopts, or what screening method a library employs. For example, phage libraries use a panning protocol in which immobilized antigens are coated on a solid surface, while yeast libraries use a flow cytometry sorting protocol with free antigens in solution (see [START_REF] Griffiths | Isolation of high affinity human antibodies directly from large synthetic repertoires[END_REF] and [START_REF] Feldhaus | Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library[END_REF] for detailed method descriptions).

Meanwhile, it also indicates that the initial screened scFvs from these naïve antibody libraries may need to be engineered further to improve their affinities for the natively unfolded protein α-syn, or to increase their thermal stabilities and antigen-binding specificities.

Antigens used for screening scFv libraries: α-Syn contains an N-terminal amphipathic region, a central hydrophobic region known as the nonamyloid component (NAC, aa 61-95), and a C-terminal acidic tail. It has been reported that the NAC region is the core sequence responsible for α-syn aggregation [START_REF] Giasson | A hydrophobic stretch of 12 amino acid residues in the middle of alpha-synuclein is essential for filament assembly[END_REF][START_REF] Bodles | Identification of the region of non-Abeta component (NAC) of Alzheimer's disease amyloid responsible for its aggregation and toxicity[END_REF]. The N-terminal sequence has a propensity to bind with acidic lipids whereas the C-terminal region appears to be involved in α-syn aggregation [START_REF] Davidson | Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes[END_REF][START_REF] Kim | Structural and functional implications of C-terminal regions of alpha-synuclein[END_REF]. Currently, the accurate molecular mechanisms of α-syn self-binding and the formation of stable oligomers and fibrils are unclear. Therefore, there are no clear strategies for predicting antigenic epitopes to produce anti-aggregation scFvs, but to explore all of the possibilities. That is to use α-syn peptides, α-syn monomers, and α-syn conformers (including α-syn oligomeric aggregates and fibrils) as antigens.

With these antigens, it is hopeful that scFvs may be generated against two kinds of potentially useful epitopes to inhibit α-syn aggregation. One type of useful epitope is the linear α-syn sequences that are the self-binding sites of two or more α-syn monomers. Among scFvs that can bind to these sequences, it is reasonable to select those that can block the self-binding sites and thus inhibit α-syn oligomer formation. The identified sequences in the

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NAC region (aa 68-76 or aa 71-82), which are required for α-syn aggregation, are the most promising candidates in this aspect. Indeed, scFv NAC32 is selected against a peptide containing the amino acids 53-87 of α-syn.

When tested in mammalian cells as an intrabody, it inhibited α-syn aggregation and reduced the cytotoxicity caused by mutant α-syn. However, we cannot exclude other N-terminal or C-terminal sequences as useful antigenic epitopes, since we do not know how these two regions play their roles in α-syn aggregation. Another type of useful epitope is the three-dimensional structures that are important for aggregation formation. ScFvs against this kind of epitope are also expected to block α-syn self-aggregation. For example, scFv D10 recognizes a conformational epitope, inhibits the formation of a high molecular weight α-syn species and rescues impaired cell adhesion caused by overexpressed α-syn. For these available anti-α-syn scFvs, a detailed description of the relationship between the epitopes and their scFvs' capacity to inhibit α-syn aggregation can be found in Table 1.

In vitro versus in situ characterization of scFvs: As we mentioned before, antigen-binding efficiencies of an scFv between in vitro and intracellular conditions are often not consistent due to the reducing environment of the cytosol. For example, both scFv F8 and scFv D10 are selected against recombinant monomeric α-syn from a human synthetic antibody library. ScFv F8 shows high affinity (K D = ~10 -8 M) whereas scFv D10 has only low affinity (K D = ~10 -6 M). However, when expressed intracellularly, scFv F8 is very unstable whereas scFv D10 is quite stable. Surprisingly, scFv D10 is able to bind α-syn inside of mammalian cells and, further, to inhibit the formation of a high molecular weight α-syn species. The phenomenon of low-affinity scFvs being functional intrabodies might be addressed by two explanatory models of antigen-antibody interaction, the "lock-and-key"

and "induced fit" models. In the "lock-and-key" model, the structures of the antigen-binding site and its corresponding antigen are highly complementary. Therefore, only minimum conformational changes are required when the antibody binds to its antigen, yielding high affinity. On the other hand, in the "induced fit" model,

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antigens, antibodies, or both, undergo conformational adjustments to facilitate antibody-antigen binding, thus resulting in low affinity [START_REF] Braden | Protein motion and lock and key complementarity in antigen-antibody reactions[END_REF]. It is likely that due to high intracellular stability, some low affinity scFvs, such as scFv D10, can accumulate to a certain amount in the cytosol and bind their antigens through an "induced fit" mechanism. Consistent with this hypothesis, other studies also revealed that the stability is much more critical than the in vitro affinity for an scFv to be a functional intrabody [START_REF] De | Analysis of the interaction between single-chain variable fragments and their antigen in a reducing intracellular environment using the two-hybrid system[END_REF]. Therefore, while in vitro characterization is helpful for identifying scFv features, it is critical to assess the performance of scFvs inside of mammalian cells in order to generate functional intrabodies.

Intracellular characterization of intrabodies:

From the above, it is apparent that intracellular stability and "intracellular affinity" are two critical aspects for evaluating the potential of an scFv to be a functional intrabody. To evaluate intracellular stability of scFvs, the pulse-chase method is a classic way to determine protein half-lives. In general, an scFv with a half-life of over 8 hours would likely have a high intracellular steady state level in order to be a functional intrabody. Meantime, it is also important that a stable scFv can fold correctly in the cytosol to maintain its antigen-binding efficiency in a reducing environment, i.e. "intracellular affinity". Unfortunately, unlike in vitro affinity assays, such as BIAcore, a direct intracellular affinity assay is currently not available. Therefore, two intrabody binding assays have been established to test indirectly for binding efficacy between intrabodies and their antigens. One strategy is to express scFvs tagged with organelle-targeting sequences to gather scFvs at certain subcellular compartments, such as nuclei. If an scFv binds to its intracellular antigen efficiently, it can convey its antigen to the same organelle. As a result, both the scFv and its antigen can be found to be enriched and colocalized in the same organelle by immunocytochemistry. For example, co-overexpression of D10-NLS (nuclear localization signal) and α-syn causes the enrichment and co-localization of both proteins in the nuclei [START_REF] Zhou | A human single-chain Fv intrabody blocks aberrant cellular effects of overexpressed alpha-synuclein[END_REF]. Of note, in scFv/antigen co-transfection experiments, the degree of accumulation of the scFv/antigen to a

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ACCEPTED MANUSCRIPT [START_REF] Begent | Clinical evidence of efficient tumor targeting based on single-chain Fv antibody selected from a combinatorial library[END_REF] certain organelle varies from cell to cell. Thus, a strict quantification of the percentage and degree of the colocalization would be helpful. For this, one can use subcellular fractionation with Western blotting to assess the colocalization efficacy, which can be quantified with image quantification softwares, such as Scion Image and NIH Image. Another method to show the binding between intrabodies and their antigens is the co-immunoprecipitation assay. In this case, intrabodies and their antigens will be considered just as two proteins overexpressed intracellularly. Co-immunnoprecipitation is used to evaluate whether or not intrabodies and their antigens bind to each other inside of cells. For instance, the D10 intrabody shows dose-dependent co-immunoprecipition with α-syn [START_REF] Zhou | A human single-chain Fv intrabody blocks aberrant cellular effects of overexpressed alpha-synuclein[END_REF]. In general, when the above two strategies are applied, they may reveal whether an scFv can bind its antigen effectively as an intrabody. For the future, a direct and real-time binding assay is needed to assess the "intracellular affinity" of intrabodies in a more accurate way so that an "intracellular K D " and a retention time can be given. Such elegant intracellular quantification assays will improve significantly the evaluation of intrabody characters.

Evaluation of anti-α-syn intrabody protective effects:

Once efficient anti-α-syn intrabodies have been identified, it is critical to evaluate whether or not these intrabodies are protective. Given that the physiological function of α-syn is still unclear, it is necessary to test the protective effect of intrabodies on both of the inhibition of α-syn aggregation and rescue from cytotoxicity caused by α-syn, which model the pathogenic hallmarks of PD ---Lewy body and neuron death. The thioflavin T aggregation assay and atomic force microscope (AFM) imaging are widely used for in vitro study of α-syn aggregation. Due to its aggregation-prone feature, α-syn forms self-aggregates in common solvents, such as phosphate buffers. By monitoring thioflavin T fluorescence intensities, the kinetics of α-syn aggregation can be established which allows one to evaluate whether or not an intrabody inhibits α-syn aggregation [START_REF] Conway | Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease[END_REF]. The AFM analysis, another in vitro tool, is often used to observe α-syn aggregates directly, allowing identification of various α-syn aggregation stages, from small aggregates to filaments [START_REF] Conway | Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease[END_REF][START_REF] Dyson | Intrinsically unstructured proteins and their functions[END_REF]. At the cellular level, except for analyzing α-syn by immunoblotting [START_REF] Zhou | A human single-chain Fv intrabody blocks aberrant cellular effects of overexpressed alpha-synuclein[END_REF], fluorescence-tagged α-syn may be used to model α-syn aggregation. For instance, the α-syn-GFP fusion protein has been used to model α-syn aggregation and its abnormal effects in yeast [START_REF] Outeiro | Yeast cells provide insight into alpha-synuclein biology and pathobiology[END_REF]. Similarly, when a fusion protein of α-syn-red fluorescent protein (RFP) is overexpressed in human neuroblastoma SH-SY5Y cells, we observed that the majority of overexpressed α-syn-RFP formed perinuclear aggregates that are both α-syn and RFP positive (Fig. 4A). Furthermore, fluorescent immunoblotting shows that the majority of overexpressed α-syn-RFP is full-length protein (Fig. 4B), thus supporting α-syn-RFP overexpression as a model of α-syn aggregation in mammalian cells. Recently, cytotoxicity assays using ST14A neuronal cells have been utilized to test the protective effect of intrabodies against α-syn toxicity. Along this line, other established α-syn cellular models, such as inducible α-syn overexpression in PC12 cells [START_REF] Tanaka | Inducible expression of mutant alpha-synuclein decreases proteasome activity and increases sensitivity to mitochondria-dependent apoptosis[END_REF] and overexpression of α-syn in primary cultured dopaminergic neurons [START_REF] Zhou | Overexpression of human alpha-synuclein causes dopamine neuron death in rat primary culture and immortalized mesencephalon-derived cells[END_REF], may also be useful for the evaluation of the key issue ---the protective effects of anti-α-syn intrabodies against neuronal death. Perhaps, the more challenging tests are whether anti-α-syn intrabodies can perform protective functions in vivo, especially in α-syn models in mice [START_REF] Giasson | Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein[END_REF], Drosophila [START_REF] Feany | A Drosophila model of Parkinson's disease[END_REF], Caenorhabditis elegans [START_REF] Lakso | Dopaminergic neuronal loss and motor deficits in Caenorhabditis elegans overexpressing human alpha-synuclein[END_REF],

etc.

Intrabody optimization: It is worth pointing out that after the initial identification of functional intrabodies that can bind to their antigens intracellularly, certain optimization strategies can be applied to obtain robust intracellular stability and "intracellular affinity" (Fig. 2). For example, the affinity of scFvs derived from naïve antibody libraries often can be further increased by random mutagenesis. Such mutagenesis can create sub-libraries with ~10 6 mutated scFvs. If sub-libraries are screened against the same antigen, a set of scFvs with higher affinities can be obtained [START_REF] Pavoni | Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein[END_REF]. Recently, it has been shown that using yeast scFv display, scFvs with high

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thermal stability can also be achieved by mutagenesis [START_REF] Weaver-Feldhaus | Directed evolution for the development of conformation-specific affinity reagents using yeast display[END_REF]. With these further steps, intrabodies with optimized intracellular stability and affinity can be achieved eventually. Such robust intrabodies should be ready for further in vivo applications, including clinical trials.

Summary: As discussed above, anti-α-syn scFvs/intrabodies have been generated and tested for their potential protective effects against α-syn malfunction. While these intrabodies show promising protective effects, more extensive studies are needed in the following two areas. First, robust intrabodies with optimized intracellular stability and "intracellular affinity" are needed in order to make intrabody as an intracellular "magic bullet". Currently, only two anti-α-syn scFvs, D10 and NAC32, have shown promising effects as intrabodies in inhibiting α-syn aggregation and in rescuing α-syn toxicity. While both scFvs show relative intracellular stability, they all have low affinity. Importantly, the production of anti-α-syn scFvs with high "intracellular affinity" is more likely to provide robust intrabodies as possible therapeutics for PD. To achieve this goal, as we proposed, it would be helpful to establish real-time analyses to evaluate intracellular intrabody-α-syn dynamic interaction.

That way, the intracellular binding efficacy of anti-α-syn intrabodies can be quantified and further used to generate high "intracellular affinity" through mutagenesis. Currently, since such "intracellular affinity assays" are unavailable, it may be important to generate the scFvs that have both intracellular stability and in vitro high affinity, with the assumption that given the intracellular stability, the in vitro affinity may reflect a certain degree of "intracellular affinity". A strategy for generating high affinity scFvs with high intracellular stability is given in Fig. 2.

In addition, the specificity of anti-α-syn intrabodies for other synuclein family proteins, such as β-syn and γ-syn, need to be tested. For instance, the D10-NLS construct failed to convey β-syn to the nucleus, thus indicating its relative specificity for α-syn inside cells. This specificity issue also applies to other available anti-α-syn scFvs listed in Table 1. The highly specific scFvs against α-syn, but not its homologous proteins, are more likely to avoid

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disturbing other normal cellular functions. Second, the current studies of anti-α-syn intrabodies are pioneer work for demonstrating proof of principle. To generate valuable anti-α-syn intrabodies as therapeutics for PD, in-depth studies are needed to answer the following questions: how intrabodies inhibit α-syn aggregation; whether the binding of intrabodies with α-syn can change α-syn degradation; whether or not and how intrabodies can provide significant protection against dopaminergic neuron death; etc. Furthermore, to overcome the intrinsic limitation of studies using cellular models, in vivo intrabody effects on α-syn pathology could provide critical insight into the potential of intrabody therapy for PD.

LRRK2

In the following, we will discuss briefly a possible use of intrabodies as potential therapeutics against cellular toxicity caused by LRRK2. Recently, the importance of LRRK2 in PD has been highlighted by the fact that LRRK2 is currently the most frequent genetic cause of all PD cases.

LRRK2 is a large complex protein of 2,527 amino acids. It contains several predicted functional domains:

Roc (Ras in complex proteins), COR domain (C-terminal of ROC), a leucine-rich repeat (LRR), a protein kinase domain, a WD40 domain and an ankryin domain. The ROC domain has GTPase activity. Currently, the functions of some of these domains, such as Roc, LRRs, Ankyrin, and WD40 domains, are unknown.

Certain PD-linked LRRK2 mutations, such as G2019S and I2020T, increase LRRK2 kinase activity, thus leading to neuronal toxicity [START_REF] West | Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity[END_REF][START_REF] Smith | Kinase activity of mutant LRRK2 mediates neuronal toxicity[END_REF][START_REF] West | Parkinson's disease-associated mutations in LRRK2 link enhanced GTP-binding and kinase activities to neuronal toxicity[END_REF]. Accordingly, abolishing the kinase activity attenuates cytotoxicity caused by mutant LRRK2, confirming that the kinase activity is likely a prerequisite for the toxic effects of mutant LRRK2 [START_REF] Greggio | Kinase activity is required for the toxic effects of mutant LRRK2/dardarin[END_REF]. Therefore, blocking LRRK2 kinase activity by intrabodies could be a potential therapeutic strategy against aberrant LRRK2 toxicity, especially when using kinase domain-specific intrabodies, which do not affect the In addition, it has been shown that LRRK2 exists as a protein complex, including homodimers. LRRK2

homodimerization is dependent upon the interaction of the ROC GTPase domain and several other domains, such as the LRR and WD40 domains [START_REF] Greggio | The Parkinson Disease-associated Leucine-rich Repeat Kinase 2 (LRRK2) Is a Dimer That Undergoes Intramolecular Autophosphorylation[END_REF]. Interestingly, it has also been found that LRRK2 can form aggregates under certain circumstances. For example, overexpression of mutant LRRK2 has led to the formation of cytoplasmic aggregates or inclusions in various cell types, suggesting that LRRK2 mutations promote protein misfolding [START_REF] Greggio | Kinase activity is required for the toxic effects of mutant LRRK2/dardarin[END_REF][START_REF] Smith | Leucine-rich repeat kinase 2 (LRRK2) interacts with parkin, and mutant LRRK2 induces neuronal degeneration[END_REF][START_REF] Macleod | The familial Parkinsonism gene LRRK2 regulates neurite process morphology[END_REF]. Therefore, intrabodies that can block LRRK2 misfolding may be protective.

Except for self-binding, LRRK2 has been shown to interact with other proteins, such as parkin [START_REF] Smith | Leucine-rich repeat kinase 2 (LRRK2) interacts with parkin, and mutant LRRK2 induces neuronal degeneration[END_REF] and Rab5b [START_REF] Shin | LRRK2 regulates synaptic vesicle endocytosis[END_REF]. In addition, LRRK2 may function on cellular membranes [START_REF] West | Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity[END_REF][START_REF] Biskup | Localization of LRRK2 to membranous and vesicular structures in mammalian brain[END_REF][START_REF] Hatano | Leucine-rich repeat kinase 2 associates with lipid rafts[END_REF] or through binding to microtubules [START_REF] Gandhi | The Roc domain of leucine-rich repeat kinase 2 is sufficient for interaction with microtubules[END_REF]. With the function and toxicity of LRRK2 being further elucidated, intrabodies may be useful for blocking specific molecular events required for the gain of toxicity of LRRK2.

Prospectives

Clearly, intrabodies provide a useful tool to study protein function intracellularly and, more importantly, have the protential to serve as therapeutics for PD. Further development of intrabody technology, such as new approaches to generating robust intracellular stability and high "intracellular affinity", novel intrabody-effector fusion proteins, organelle-specific intrabodies, and protein domain-specific intrabodies, will maximize intrabody therapeutic efficacy. In-depth studies of the intrabody characters and their anti-PD effects and mechanisms in various PD models, from in vitro to in vivo, will provide solid evidence for exploring the potential of intrabody therapeutic application. The development of gene delivery methods, which are the means of expressing intrabody in target cells, will increase the in vivo therapeutic efficiency of intrabodies. In addition, since the

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studies on the molecular mechanisms of PD pathology are advancing quickly, newly identified "toxic" molecules will provide additional targets for the intrabody therapy. Together, multi-field technological and knowledge advances related to the intrabody therapy hold one of the promises for the cure of PD. and further optimization of intracellular stability and affinity. This approach applies with various antibody display system, including phage display and yeast display antibody technologies, toward yielding robust intrabodies as therapeutics.

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Figure 3. A central molecular hypothesis for the intrabody therapy against the α-syn phathology. By blocking α-syn self-aggregation, intrabodies restore the balance between the natively unfolded structure of α-syn and its ordered philological structures, rendering therapeutic effects. (a.a.) α-syn (K D , M) aggregates F8 [49] Phage 101-111/27-37 ? 10 -8 Inhibit aggregation Unstable expression D10 [25] Phage * yes 10 -6 ? Inhibit α-syn aggregation and rescue impaired adhesion 6E [50] Phage * yes ? ? ? 3 [52] Phage 71-85 yes ? ? ? 14 [52] Phage 106-120 yes ? ? ? 15 [52] Phage 117-131 yes ? ? ? D5 [51] Phage * yes ? Inhibit aggregation ? NAC3 [26] Yeast 61-78 ? ? ? No significant protection NAC24 [26] Yeast 61-78 ? ? ? Unstable expression and no significant protection NAC32 [26]

12 various

 12 cellular pathways. While α-syn may utilize its unfolded structure to adopt several possible ordered structures to function, this possible biological benefit comes with the following potential downside: α-syn can form a stable β-sheet structure by self-binding under certain conditions. The latter include increased α-syn protein levels exceeding a threshold, genetic mutations which cause potency for self-oligomerization, changes in physiological conditions including oxidative stress, and post-translational modifications including dopamine-α-syn adducts. As a result, when the protein quality control system of a cell becomes impaired, the physiological balance that favors the formation of beneficial α-syn structures may abnormally favor α-syn self-aggregation. Such self-aggregation may in turn underlie the formation of α-syn aggregates, fibrils and LBs.

  D M A N U S C R I P T ACCEPTED MANUSCRIPT 27 kinase domain of mitochondrial PINK1 faces the cytoplasm, Proc. Natl. Acad. Sci. U. S. A 105 (2008) 12022-12027.

Figure 1 .

 1 Figure 1. Schematic representation of antibody, scFv, and intrabody molecules. A typical antibody structure

Figure 2 .

 2 Figure 2. Schematic representation of the production of scFvs of interest, identification of functional intrabodies,

Figure 4 .

 4 Figure 4. An α-syn aggregation model in human neuroblastoma SH-SY5Y cells. (A) While cells expressing RFP

  reported. *: The scFvs D10, 6E, and D5 recognize α-syn conformational epitopes, although the amino acid sequences that constitute the epitopes are not identified. A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

Table 1 .

 1 Published scFvs against α-syn

	Name	Source	Epitope	Binding to. Affinity In vitro effects	Intracellular effects
		library

Acknowledgements

. We thank Drs. Anne Messer (Wadsworth Center, New York State Department of Health) and Howard Federoff (Georgetown University) for their insightful comments on the manuscript. The authors are supported by grants from the US National Institutes of Health (NS062180, NS11766-27A1, AG21617-01A1, NS38370-09), the US Department of Defense (DAMD 17-03-1-02), the Parkinson's Disease Foundation, the Hartman Foundation, the Muscular Dystrophy Association/WOW.


Christopher D Morrison

Peter Huypens

Laura K Stewart

Thomas W Gettys

IMPLICATIONS OF CROSSTALK BETWEEN LEPTIN AND INSULIN SIGNALING DURING THE DEVELOPMENT OF DIET INDUCED OBESITY

Insulin and leptin play complementary roles in regulating the consumption, uptake, oxidation and storage of nutrients. Chronic consumption of diets that contain a high proportion of calories from saturated fat induces a progressive deterioration in function of both hormones. Certain rat lines and strains of mice are particularly sensitive to the obesogenic and diabetogenic effects of high fat diets, and have been used extensively to study the developmental progression of insulin and leptin resistance in relation to the increasing adiposity that is characteristic of their response to these diets. Some aspects of the diminished efficacy of each hormone are secondary to increased adiposity but a consensus is emerging to support the view that direct effects of dietary components or their metabolites, independent of the resulting obesity, play important roles in development of insulin and leptin resistance. In this minireview, we will examine the implications of crosstalk between leptin and insulin signaling during the development of diet-induced obesity, emphasizing potential interactions between pathways that occur among target sites, and exploring how these interactions may influence the progression of obesity and diabetes.

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Introduction

After the original description of variable diabetogenic effects of high fat diets among mouse strains by Surwit et al. [1; 2], the fat-sensitive C57BL/6J mouse emerged as a key model to study the developmental pathology of the obese/diabetic syndrome that develops after chronic consumption of high fat (HF) diets. Various rat lines respond similarly, and studies contrasting rats or mice on LF versus HF diets have illustrated the global and tissue-specific changes that occur during the time frame when defects in leptin and insulin sensitivity first become apparent. However, the question of whether the initial stages of glucose intolerance are the product of a uniform progression of IR across tissues or the result of a sequential but punctuated progression of IR among tissues remains open. HF diets also increase circulating leptin levels and promote the development of leptin resistance.

Potential mechanisms of diet-induced leptin resistance include decreased access of leptin to hypothalamic targets, diminished responsiveness of hypothalamic neurons to leptin, modified translation of leptin-dependent signals in downstream target tissues, or some combination thereof [START_REF] Myers | Mechanisms of leptin action and leptin resistance[END_REF].

The complex progression of events involved in the development of insulin and leptin resistance represents a significant impediment to understanding the underlying cause and effect mechanisms within each pathway. Given the overlap within the respective signaling pathways of each hormone, crosstalk between the pathways is likely to become significant and contribute to the progression of diet-induced obesity and diabetes.

I. Developmental Progression of Insulin and Leptin Resistance During Diet-Induced Obesity

The strong relationship between obesity and diabetes is well documented but the underlying mechanisms which link development of obesity to initiation and progression of insulin and leptin resistance are less well understood. This is in part due to the chronic nature of both conditions and the associated progressive deterioration in function of multiple, highly integrated organ systems which function in concert to maintain glucose and energy homeostasis. Because of the temporal nature of the process, it has been difficult to identify the tissue sites and events which constitute the initial steps

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of IR and link deterioration in insulin sensitivity to progression of diet-induced obesity and diabetes.

For example, without careful design, it is difficult to establish that observed changes in tissue responses and function are not secondary to the initial events which alter insulin sensitivity, and thus a consequence rather than an underlying cause of IR.

The original experiments of Surwit et al. [1; 2] involved weaning male C57BL/6J mice onto a diet high in saturated fat (58 kcal%) derived primarily from coconut oil, and the mice developed fasting hyperglycemia and hyperinsulinemia after 6 wks on the diet. Variations of this initial design have used different amounts (58 vs 45 kcal%) and sources of fat (lard vs coconut oil) in the diet, as well as age when the diet was first introduced. The modified designs have also been used with various rat lines, and, in general, the responses have been similar in the sense that the animals increased fat deposition and developed some degree of IR, depending on length of time on the diet. Relatively few of the many studies in rats and mice have used longitudinal approaches coupled with in vivo measurements (hyperinsulinemic-euglycemic clamps) to evaluate whole body insulin sensitivity and identify the tissue sites where IR first appears. However, the consensus emerging from limited studies with young animals is that the very high fat (55-60 kcal%) diets produce a rapid deterioration (3 wks) in whole body insulin sensitivity that coincides with a simultaneous reduction in insulin-dependent glucose uptake in peripheral tissues coupled with a failure of insulin to suppress hepatic glucose production [START_REF] Park | Unraveling the temporal pattern of diet-induced insulin resistance in individual organs and cardiac dysfunction in C57BL/6 mice[END_REF][START_REF] Storlien | Fat feeding causes widespread in vivo insulin resistance, decreased energy expenditure, and obesity in rats[END_REF][START_REF] Kraegen | In vivo insulin resistance in individual peripheral tissues of the high fat fed rat: assessment by euglycaemic clamp plus deoxyglucose administration[END_REF]. The response pattern is common to both rats and mice on the very high fat diet formulation (55-60 kcal%) and supports the view that whole body IR is the product of a rapid, uniform progression of IR among peripheral tissues [START_REF] Park | Unraveling the temporal pattern of diet-induced insulin resistance in individual organs and cardiac dysfunction in C57BL/6 mice[END_REF][START_REF] Storlien | Fat feeding causes widespread in vivo insulin resistance, decreased energy expenditure, and obesity in rats[END_REF][START_REF] Kraegen | In vivo insulin resistance in individual peripheral tissues of the high fat fed rat: assessment by euglycaemic clamp plus deoxyglucose administration[END_REF][START_REF] Oakes | Mechanisms of liver and muscle insulin resistance induced by chronic high-fat feeding[END_REF]. In contrast, a different outcome is obtained when rats or mice are given HF diets with a lower proportion of calories from fat (ie 45 kcal% versus 55-60 kcal%) . Consuming the 45 kcal% HF diet for 1-5 wks resulted in loss of suppression of hepatic glucose production by insulin without any change in insulin-sensitive glucose uptake in peripheral tissue [8; 9]. These findings argue that loss of insulin sensitivity in liver is responsible for the initial deterioration in whole body insulin sensitivity. Mice on the 45 kcal% HF diet for longer periods (12-14 wks) develop IR in peripheral tissues [START_REF] Dhalla | A1 adenosine receptor partial agonist lowers plasma FFA and improves insulin resistance induced by high-fat diet in rodents[END_REF], but this outcome may be a reflection of longer term consumption of the 45 kcal% HF diet and indicative of later stages in the progression. It seems likely

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that the reported differences among studies are due to a combination of differences in amount, source, and saturation of dietary fat, as well as length of exposure and age of animals when the test diets were first introduced.

Leptin is a key component of the neuroendocrine circuitry that regulates food intake and energy utilization, and its absence in ob/ob mice produces a complex metabolic syndrome characterized by hyperphagia and lowered rates of energy expenditure [START_REF] Zhang | Positional cloning of the mouse obese gene and its human homologue[END_REF][START_REF] Friedman | The function of leptin in nutrition, weight, and physiology[END_REF][START_REF] Gettys | Encylopedia of Biological Chemistry[END_REF]. However, dietinduced obesity is not associated with an absence of leptin, as circulating leptin is actually increased in proportion to the increase in body adiposity. The failure of this hyperleptinemia to produce any compensatory decrease in food intake or body weight was initially viewed as evidence of leptin resistance. The existence of high fat diet-induced leptin resistance has since been directly demonstrated as a failure of exogenous leptin to suppress food intake or induce weight loss [START_REF] Maffei | Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects[END_REF][START_REF] Considine | Serum immunoreactive-leptin concentrations in normalweight and obese humans[END_REF][START_REF] Halaas | Physiological response to long-term peripheral and central leptin infusion in lean and obese mice[END_REF].

Chronic consumption of HF diets promotes the development of leptin resistance as defined by both criteria, but understanding the temporal sequence of events involved in the development of dietinduced leptin resistance is complicated by the anatomical and functional complexity of the leptin sensing, signaling, and effector systems. Leptin functions to maintain body weight homeostasis by modulating appetite control centers in the hypothalamus and energy utilization in part by increasing sympathetic outflow to peripheral tissues [START_REF] Commins | Norepinephrine is required for leptin effects on gene expression in brown and white adipose tissue[END_REF][START_REF] Collins | Role of leptin in fat regulation[END_REF][START_REF] Haynes | Receptor-mediated regional sympathetic nerve activation by leptin[END_REF][START_REF] Scarpace | Leptin induction of UCP1 gene expression is dependent on sympathetic innervation[END_REF]. Sympathetic outflow to adipose tissue enhances fat oxidation by coordinated transcriptional activation of genes favoring substrate oxidation [21; 22].

Thus in the broadest sense, leptin acts to regulate adipose tissue reserves through a coordinated suppression of energy intake and stimulation of fat oxidation. The consensus is that leptin initiates these signals by crossing the blood brain barrier and binding to receptors on hypothalamic NPY/AGRP and POMC neurons, which activate STAT-3 through a Jak2-dependent pathway [START_REF] Vaisse | Leptin activation of Stat3 in the hypothalamus of wildtype and ob/ob mice but not db/db mice[END_REF][START_REF] Tartaglia | Identification and expression cloning of a leptin receptor[END_REF][START_REF] Cowley | Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus[END_REF][START_REF] Bjorbaek | Divergent signaling capacities of the long and short isoforms of the leptin receptor[END_REF].

Leptin receptors are expressed on neurons located primarily but not exclusively within the arcuate nuclei, but emerging evidence suggests that proper integration of the complex behavioral and metabolic responses to leptin requires input from these additional populations of leptin responsive neurons [3; 27].

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Studies examining leptin sensitivity have typically measured the ability of exogenous leptin, given either peripherally or centrally, to activate hypothalamic STAT-3, reduce food intake, reduce body weight, or increase sympathetic nervous system (SNS) activity. In C57BL/6J mice weaned onto a 45 kcal% HF diet, Van Heek et al. [START_REF] Van Heek | Diet-induced obese mice develop peripheral, but not central, resistance to leptin[END_REF] reported that the anorectic response to leptin was lost after 16 d on the HF diet. Using essentially the same experimental design, El Haschimi et al. [START_REF] El-Haschimi | Two defects contribute to hypothalamic leptin resistance in mice with diet-induced obesity[END_REF] found that the 45 kcal% HF diet did not alter the ability of peripherally injected leptin to activate hypothalamic STAT3. In contrast, C57BL/6J mice weaned onto a higher percentage of dietary fat (58 kcal% vs 45 kcal%) became fully resistant to both the anorectic and metabolic effects of peripherally injected leptin after 4 wks on the diet [START_REF] Prpic | Differential mechanisms and development of leptin resistance in A/J vs C57BL/6J mice during diet-induced obesity[END_REF]. However, the responses to leptin were not compromised when it was administered centrally [START_REF] Prpic | Differential mechanisms and development of leptin resistance in A/J vs C57BL/6J mice during diet-induced obesity[END_REF], suggesting compromised access to hypothalamic target neurons as a basis for resistance to peripherally injected leptin [31; 32]. Although working with a different strain of mice (AKR/J), Van Heek et al [START_REF] Van Heek | Diet-induced obese mice develop peripheral, but not central, resistance to leptin[END_REF] proposed a similar mechanism to explain resistance to peripheral leptin after 8 wks on a HF diet. Returning to the C57BL/6J model, longer term consumption (15 wks) of the 45 kcal% HF diet fully compromised the ability of peripherally injected leptin to activate STAT3, but also reduced the capacity of hypothalamic neurons to respond to centrally injected leptin [START_REF] El-Haschimi | Two defects contribute to hypothalamic leptin resistance in mice with diet-induced obesity[END_REF]. The diminished response to central leptin argues that the HF diet produced a second defect compromising cellular signaling at some point upstream of STAT3 [START_REF] El-Haschimi | Two defects contribute to hypothalamic leptin resistance in mice with diet-induced obesity[END_REF].

An important component of leptin's metabolic effects is mediated through SNS stimulation of fat oxidation in adipose tissue [21; 22]. Therefore, the observed decrease in β-adrenergic receptor function in adipose tissue after long term (16 wks) consumption of the 58 kcal% HF diet [START_REF] Collins | Strain-specific response to β3-adrenergic receptor agonist treatment of diet-induced obesity in mice[END_REF] could diminish efficacy of SNS input and limit translation of this component of leptin's actions. However, in recent studies with C57BL/6J mice consuming the 58 kcal% HF diet for 8 wks [START_REF] Prpic | Differential mechanisms and development of leptin resistance in A/J vs C57BL/6J mice during diet-induced obesity[END_REF], centrally administered leptin activated hypothalamic STAT3, reduced food consumption, and increased UCP1 mRNA in BAT. The latter response indicates that leptin signaling to brown adipose tissue was intact, but in the same mice leptin was unable to increase UCP1 expression in retroperitoneal WAT but was able to decrease leptin mRNA. Given that both WAT responses are mediated by SNS input [17; 34],

the intactness of the latter response indicates that SNS-dependent β-adrenergic activation was not

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compromised in retroperitoneal WAT. Additional studies showed that the defect in UCP1 induction induced by the HF diet was downstream of β-adrenergic receptor activation in retroperitoneal WAT [START_REF] Prpic | Differential mechanisms and development of leptin resistance in A/J vs C57BL/6J mice during diet-induced obesity[END_REF]. Together, these studies illustrate how HF diets can induce leptin resistance in part by compromising the ability of peripheral target tissues to translate input from central effector systems (SNS) that are regulated by hypothalamic leptin signaling.

Lastly, recent work has shown that the time course of leptin signaling after peripheral injection of leptin is not synchronized within all regions of hypothalamic neurons expressing leptin receptors, whereas centrally administered leptin produces simultaneous leptin-dependent signaling among these neuronal populations [27; 35; 36]. This finding argues that peripherally injected leptin does not reach all leptin responsive neurons at the same time, and it suggests that a population of ARC neurons expressing leptin receptors may extend processes across the BBB to directly contact the circulation [START_REF] Faouzi | Differential accessibility of circulating leptin to individual hypothalamic sites[END_REF]. These findings also raise the interesting possibility that this population of leptin responsive neurons may be more subject to desensitization by high leptin levels or other circulating factors associated with chronic consumption of HF diets [START_REF] Banks | Triglycerides induce leptin resistance at the blood-brain barrier[END_REF].

II. Mechanisms of Diet-Induced Insulin Resistance

A growing consensus supports the view that accumulation of lipid in tissues not designed for storage is a key initial step in the progression towards insulin resistance (IR) [37; 38]. Lipodystrophy is characterized by defective storage of triglyceride in adipose tissue, and the resulting oversupply of lipids to metabolic organs produces ectopic fat accumulation and the development of IR [START_REF] Kim | Mechanism of insulin resistance in A-ZIP/F-1 fatless mice[END_REF].

Chronic consumption of HF diets results in a similar oversupply of lipids to metabolic organs that is temporally related to periods of rapid weight gain, and associated with decreased insulin sensitivity and impaired glucose homeostasis [38; 40]. Muoio et al [41; 42] have presented evidence that the oversupply of lipids to mitochondria produces a mismatch between TCA cycle flux and fat oxidation that results in depletion of organic acid intermediates, incomplete fatty acid oxidation, and accumulation of lipotoxic short chain fatty acylcarnitines. Although the underlying mechanisms remain unclear, the accumulation of these lipotoxic metabolites compromises insulin signaling and is

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associated with the onset of IR in skeletal muscle of HF fed Wistar rats. A reversal of the mismatched metabolic flux could be obtained by increasing mitochondrial oxidative capacity or limiting entry of fatty acids into mitochondria [START_REF] Muoio | Hepatic expression of malonyl-CoA decarboxylase reverses muscle, liver and wholeanimal insulin resistance[END_REF][START_REF] Koves | Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle insulin resistance[END_REF][START_REF] Koves | PPARgamma coactivator-1alpha -mediated metabolic remodeling of skeletal myocytes mimics exercise training and reverses lipid-induced mitochondrial inefficiency[END_REF], both of which restored complete fatty acid oxidation and insulin sensivity. It remains to be seen whether a similar mechanism is involved in compromising insulin sensitivity in other peripheral tissues such as the liver, where IR first appears with the 45 kcal% HF diet.

III. Cellular Mechanisms of Diet-Induced Leptin Resistance

While the existence of leptin resistance is widely accepted, the physiological and cellular mechanisms that underlie this resistance are less clear. This is due in part to the complexity of the system itself, and the fact that leptin resistance could occur at multiple points within this multifaceted cascade. It has been argued that high fat diets lead to reduced leptin transport across the blood brain barrier (BBB) [START_REF] Banks | Impaired transport of leptin across the blood-brain barrier in obesity is acquired and reversible[END_REF], consistent with work demonstrating reduced sensitivity to peripherally administered leptin prior to loss of central leptin sensitivity [28; 30]. Nevertheless, the cellular mechanisms of leptin transport and its disruption in obesity are not fully resolved [31; 32; 44; 45], and some neurons within the hypothalamic arcuate nucleus may extend projections beyond the BBB to directly sample hormones in circulation [START_REF] Faouzi | Differential accessibility of circulating leptin to individual hypothalamic sites[END_REF]. It has also been established that HF diets compromise leptin signaling within target neurons [29; 46-48]. This resistance is manifest as a loss of leptindependent suppression of food intake and impaired activation of specific signaling pathways, namely Stat3 and PI3K [3; 47]. Leptin resistance could also occur indirectly, with loss of sensitivity in various effector pathways mediating downstream effects of the leptin receptor. For instance, some have detected a HF-induced loss of sensitivity to melanocortins, although others fail to detect this resistance [START_REF] Chandler | Feeding response to melanocortin agonist predicts preference for and obesity from a high-fat diet[END_REF][START_REF] Clegg | Increased dietary fat attenuates the anorexic effects of intracerebroventricular injections of MTII[END_REF][START_REF] Lu | Leptin resistance in obesity is characterized by decreased sensitivity to proopiomelanocortin products[END_REF][START_REF] Pierroz | Effects of acute and chronic administration of the melanocortin agonist MTII in mice with diet-induced obesity[END_REF]. Lastly, there is evidence for selective leptin resistance based on physiological endpoint, a loss of leptin-dependent suppression of food intake despite a maintenance of leptindependent stimulation of sympathetic outflow to peripheral tissues [30; 53; 54]. In summary, these examples illustrate the challenges that are inherent to understanding the origins and mechanisms of leptin resistance.

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In assessing potential causes of leptin resistance, perhaps the most progress has been made in defining molecular mediators of cellular leptin resistance. This work has identified two molecules which inhibit leptin receptor signaling; suppressor of cytokine signaling 3 (Socs3) and protein tyrosine phosphatase 1B (PTP1B). Socs3 is one of a family of proteins produced in response to cytokine signaling which functions as an intracellular negative feedback signal [START_REF] Howard | Attenuation of leptin and insulin signaling by SOCS proteins[END_REF]. Socs3 expression is induced by Stat3 signaling and binds to the leptin receptor, blocking Stat3 activation [56; 57]. Mice bearing genetic modifications which delete Socs3 or inhibit its ability to bind the leptin receptor exhibit reduced food intake and body weight and are resistant to diet induced obesity [START_REF] Bjornholm | Mice lacking inhibitory leptin receptor signals are lean with normal endocrine function[END_REF][START_REF] Howard | Enhanced leptin sensitivity and attenuation of diet-induced obesity in mice with haploinsufficiency of Socs3[END_REF][START_REF] Mori | Socs3 deficiency in the brain elevates leptin sensitivity and confers resistance to diet-induced obesity[END_REF].

Protein tyrosine phosphatase 1B (PTP1B) is similarly implicated in leptin resistance due to its ability to limit the magnitude and duration of leptin receptor signaling. PTP1B binds to and dephosphorylates Janus Kinase 2 (Jak2), the initial tyrosine kinase activated by the leptin receptor [61; 62]. Overexpression of PTP1B in vitro dampens signaling from the leptin receptor [START_REF] Kaszubska | Protein tyrosine phosphatase 1B negatively regulates leptin signaling in a hypothalamic cell line[END_REF], and mice genetically deficient for PTP1B are lean and hypersensitive to leptin [61; 62]. Neuron-specific deletion of PTP1B recapitulates the leptin hypersensitivity and resistance to diet-induced obesity [START_REF] Bence | Neuronal PTP1B regulates body weight, adiposity and leptin action[END_REF], and local inhibition of PTP1B via pharmacological or adenoviral approaches enhances the effects of central leptin injection [65; 66]. Thus these data indicate that PTP1B, like Socs3, tonically inhibits signaling from the leptin receptor.

Interestingly, Socs3 and PTP1B are also associated with regulation of insulin signaling. PTP1B limits insulin signaling by binding to and dephosphorylating the insulin receptor, and mice with global PTP1B deficiency are hypersensitive to insulin [START_REF] Elchebly | Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene[END_REF]. Similarly, Socs3 inhibits insulin receptor signaling in vitro and in vivo [68; 69]. Given that leptin and insulin regulate a common set of hypothalamic neurons, the overlapping function of these two inhibitors has the potential to produce significant crosstalk between the signaling pathways, particularly during diet induced obesity as hyperinsulinemia and hyperleptinemia develop [START_REF] Niswender | Insulin and leptin revisited: adiposity signals with overlapping physiological and intracellular signaling capabilities[END_REF]. Thus PTP1B and/or Socs3 could underlie the combined loss of sensitivity to leptin and insulin that are observed in obesity.

IV. Direct and Indirect Effects of High Fat Diets on Leptin SignalingAvailable evidence suggests

that high fat diets induce leptin resistance via both direct and indirect mechanisms. Direct

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mechanisms refer to the ability of high fat diets to induce leptin resistance in the absence of obesity, presumably via direct effects of dietary fat or its metabolites on leptin-sensitive neurons. In contrast, indirect mechanisms reflect an induction of leptin resistance that is secondary to obesity and the associated hyperleptinemia and are thus not dependent on the presence of excess dietary fat.

Several lines of evidence indicate that HF diets can induce hypothalamic leptin (and insulin) resistance in the absence of either obesity or elevated circulating leptin. A rapid diet-induced leptin and insulin resistance has been detected prior to any change in body adiposity [START_REF] Wang | Overfeeding rapidly induces leptin and insulin resistance[END_REF], while others have provided evidence for central insulin resistance in HF-fed animals, even when obesity is prevented via caloric restriction [START_REF] Woods | Consumption of a high-fat diet alters the homeostatic regulation of energy balance[END_REF]. Obesity prone rat lines exhibit reduced central leptin and insulin sensitivity prior to becoming obese on HF diets [START_REF] Clegg | Reduced anorexic effects of insulin in obesity-prone rats fed a moderate-fat diet[END_REF][START_REF] Levin | Obesity-prone rats have normal blood-brain barrier transport but defective central leptin signaling before obesity onset[END_REF][START_REF] Levin | F-DIO obesity-prone rat is insulin resistant before obesity onset[END_REF], suggesting that some degree of basal resistance contributes to their propensity to become obese. We recently examined leptin signaling in ob/ob mice to determine whether HF diets could modify leptin sensitivity in the absence of leptin.

Notwithstanding the high initial sensitivity of ob/ob mice to exogenous leptin (see below), consumption of the HF diet for 14 days attenuated leptin's anorectic effects, indicating that leptin resistance can be induced in the absence of leptin and hyperleptinemia [START_REF] White | Increased hypothalamic PTP1B in response to diet-induced obesity and chronic hyperleptinemia[END_REF]. Lastly, these findings are also consistent with evidence that hypothalamic neurons are sensitive to and respond directly to fatty acids and other lipid metabolites ([77; 78] and see section below).

In addition to direct effects of dietary fat on leptin signaling efficacy, the evidence also supports mechanisms that are indirect and secondary to the development of obesity and chronic hyperleptinemia. Nearly all settings of leptin resistance are associated with a chronic elevation of circulating leptin, and it appears that chronic hyperleptinemia inhibits leptin sensitivity via the down regulation of leptin receptors and the stimulation of negative feedback molecules such as Socs3 and PTP1B [56; 68; 79]. Additional evidence supporting a role for hyperleptinemia stems from genetic models. Despite their massive obesity, leptin-deficient ob/ob mice are highly sensitive to leptin [START_REF] Harris | A leptin dose-response study in obese (ob/ob) and lean (+/?) mice[END_REF].

Similarly, the leptin resistance of agouti viable yellow (A y ) mice was diminished when leptin was removed by crossing the A y mutation onto the ob/ob background [START_REF] Boston | Independent and additive effects of central POMC and leptin pathways on murine obesity[END_REF]. Together these observations suggest that a component of the leptin resistance of A Y mice is induced by hyperleptinemia.

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Chronic exposure to high leptin induces a form of leptin-induced leptin resistance [48; 82], and animals chronically exposed to elevated leptin levels exhibit increased weight gain on a HF diet [83; 84]. Together, these findings argue that this leptin-induced leptin resistance alters the regulation of energy homeostasis and increases the risk for obesity.

Viewed collectively, these findings indicate that leptin resistance is both a cause and a consequence of obesity [START_REF] Scarpace | Elevated leptin: consequence or cause of obesity?[END_REF]. Loss of leptin signaling clearly predisposes to weight gain, while interventions which enhance leptin signaling protect against diet induced obesity. These observations, coupled with evidence for HF-induced, obesity independent reductions in leptin sensitivity, support a causative role for leptin resistance in the development of obesity. However, a consequence of the expanding adipose mass of obesity is a chronic increase in circulating leptin, which acts on the hypothalamus to further dampen leptin signaling.

V. Direct Effects of Fatty Acids on Hypothalamic Neurons.

In addition to responding to circulating hormones such as leptin and insulin, hypothalamic neurons also possess a nutrient sensing system that responds to circulating lipids and provides regulatory inputs into systemic glucose homeostasis. Increasing hypothalamic long chain fatty acids (LCFA-CoA) via either direct ICV administration of oleic acid [START_REF] Obici | Central administration of oleic acid inhibits glucose production and food intake[END_REF] or inhibition of hypothalamic carnitine palmitoyltransferase-1 (CPT-1) [87; 88] causes a marked decrease in hepatic glucose production via suppression of glycogenolysis. This hypothalamic lipid sensing mechanism appears to require the opening of hypothalamic KATP channels and an intact vagus nerve [START_REF] Lam | Hypothalamic sensing of circulating fatty acids is required for glucose homeostasis[END_REF]. While LCFAs suppress hepatic glucose output via the hypothalamus, they increase hepatic glucose production through a direct effect on the liver [START_REF] Lam | Free fatty acids increase basal hepatic glucose production and induce hepatic insulin resistance at different sites[END_REF]. Thus, this opposing effect may serve an autoregulatory function to balance the centrally mediated inhibitory effects of LCFA. In addition to LCFAs, there is also evidence that triglycerides may act in the brain to reduce leptin transport across the blood brain barrier [START_REF] Banks | Triglycerides induce leptin resistance at the blood-brain barrier[END_REF]. Lastly, given the substantial overlap between leptin and insulin signaling and the emerging consensus that dysregulation of lipid metabolism is at the core of peripheral insulin resistance, it appears likely that similar mechanisms occur in the brain and could contribute to central leptin resistance. Taken together, these data indicate that fatty acids, triglycerides and lipid

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molecules act locally in the brain to influence leptin transport, food intake and peripheral glucose metabolism, and as such provide additional mechanisms through which the chronic consumption of a HF diet might be compromising body weight and glucose homeostasis.

VI. Implications of Crosstalk between Leptin and Insulin During Development of Diet-Induced Obesity

Postweaning consumption of diets rich in saturated fatty acids results in the progressive development of insulin resistance, leptin resistance, and obesity in C57BL/6J mice. Maintenance of glucose and energy homeostasis requires the proper integration of complex communication and effector systems, and the collective evidence shows that chronic consumption of HF diets progressively compromises the function of essential components and steps within these systems. The difficulty in understanding the underlying sequence of events in this overall process is underscored by the fact that the progression is also dependent on the source and amount of fat in the diet.

Therefore, the following comments are an attempt to summarize the key early responses of C57BL/6J mice to HF diets, suggesting potential points of interaction between insulin and leptin signaling that may contribute to or accentuate the progressive dysregulation of glucose and energy homeostasis.

Consumption of the very high fat (58 kcal%) diet formulation for 2-3 wks after weaning produces a rapid, uniform increase in IR among peripheral tissues [4; 5]. The IR, detected by hyperinsulinemic-euglycemic clamps, is a reflection of both compromised insulin-dependent glucose uptake in peripheral tissues and compromised insulin-dependent suppression of hepatic glucose production [START_REF] Park | Unraveling the temporal pattern of diet-induced insulin resistance in individual organs and cardiac dysfunction in C57BL/6 mice[END_REF]. After 2 wks on the 58 kcal% HF diet, C57BL/6J mice are significantly fatter than LF-fed mice [START_REF] Watson | Differential regulation of leptin release and function in A/J versus C57BL/6J mice during diet-induced obesity[END_REF] and by 4 wks are completely resistant to peripherally injected leptin [30; 35]. Mice on the 58 kcal% HF diet retain sensitivity to centrally administered leptin at this time point, increasing hypothalamic STAT3 phosphorylation, reducing food intake and increasing UCP1 expression in BAT after ICV injection of leptin [START_REF] Prpic | Differential mechanisms and development of leptin resistance in A/J vs C57BL/6J mice during diet-induced obesity[END_REF]. Together these data indicate that the 58 kcal% HF formulation produces a rapid and almost simultaneous increase in adiposity and deterioration of signaling through insulin and leptin. Moreover, it appears that beyond the 4 wk time point, it becomes difficult to argue that observed changes are not secondary to signaling defects developed earlier in the progression.

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The progression of both IR and leptin resistance is significantly slower with the 45 kcal% versus the 58 kcal% HF diet. After 4 wks on the 45 kcal% formulation, activation of hypothalamic STAT3 by peripheral leptin was not diminished in C57BL/6J mice [START_REF] El-Haschimi | Two defects contribute to hypothalamic leptin resistance in mice with diet-induced obesity[END_REF], and it was only after 15 wks on the HF diet that resistance to leptin was observed. Using the 45 kcal% HF diet, recent studies have shown that ambient temperature and rearing conditions also affect the progression of diet-induced leptin resistance [START_REF] Harris | Leptin-induced changes in body composition in high fat-fed mice[END_REF][START_REF] Bowen | Method of leptin dosing, strain, and group housing influence leptin sensitivity in high-fat-fed weanling mice[END_REF][START_REF] Haltiner | Leptin action is modified by an interaction between dietary fat content and ambient temperature[END_REF]. However, it should be noted that Munzberg et al. [START_REF] Munzberg | Region-specific leptin resistance within the hypothalamus of diet-induced obese mice[END_REF] have proposed that leptin resistance may develop in a region-specific manner within the hypothalamus of diet-induced obese mice, with resistance developing initially within the arcuate nuclei. If different hypothalamic regions regulate different components of the leptin response, it could explain how the development of selective leptin resistance among subgroups of responsive nuclei could eliminate certain components of leptin's actions while others are preserved [START_REF] Carreia | The concept of selective leptin resistance: evidence from agouti yellow obese mice[END_REF].

IR develops sooner (1-3 wks) than leptin resistance in both rats [8; 9] and C57BL/6J mice [START_REF] Stewart | Failure of dietary quercetin to alter the temporal progression of insulin resistance among tissues of C57BL/6J mice during the development of diet-induced obesity[END_REF] consuming the 45 kcal% HF diet. Using hyperinsulinemic-euglycemic clamps to measure in vivo insulin sensitivity, a modest decrease in glucose utilization is detected in C57BL/6J mice after 3 wks on the HF diet. This decrease in insulin sensitivity can be fully accounted for by failure of insulin to suppress hepatic glucose production, with no decrease in sensitivity of peripheral tissues to insulin. Hepatic IR becomes somewhat more severe between 3 and 8 wks on the 45 kcal% HF diet, but peripheral tissues retain full responsiveness to insulin during this period [START_REF] Stewart | Failure of dietary quercetin to alter the temporal progression of insulin resistance among tissues of C57BL/6J mice during the development of diet-induced obesity[END_REF]. It is noteworthy that the increase in hepatic IR between 3 and 8 wks is sufficient to increase pancreatic insulin release such that fasting insulin levels are ~2 fold higher than levels in control mice on a LF diet. The establishment of hyperinsulinemia during a period when peripheral tissues retain full insulin sensitivity has important physiological implications. In addition to promoting glucose uptake in peripheral tissues, insulin also activates phosphodiesterase 3B [START_REF] Beebe | Discriminative insulin antagonism of stimulatory effects of various cAMP analogs on adipocyte lipolysis and heptocyte glycogenolysis[END_REF][START_REF] Corbin | Discriminative insulin antagonism of effects of different cAMP analogs in intact mammalian cells[END_REF][START_REF] Loten | Stimulation of a low Km phosphodiesterase from liver by insulin and glucagon[END_REF], resulting in the degradation of cyclic AMP and the antagonism of glucagon signaling in the liver. If the liver was responsive to insulin during this period, the increase in insulin would have the corrective effect of suppressing hepatic glucose production.

The hyperinsulinemia also could have important effects in adipose tissue, where the lipogenic and antilipolytic effects of insulin create conditions favoring fat deposition. We have shown that the time

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ACCEPTED MANUSCRIPT frame (3 to 8 wks) when hyperinsulinemia develops coincides with a significant increase in the rate of fat deposition. This may be a mechanism to compensate for the oversupply of lipids produced by the 45 kcal% HF diet, and effectively slow the progression of IR to peripheral tissues by preventing or delaying ectopic fat accumulation. If correct, this suggests that there may be a threshold of dietary fat that determines whether the early stage of IR is limited to the liver or extends to other peripheral tissues.

The development of hyperinsulinemia between 3 to 8 wks may also have important effects on the endocrine function of adipose tissue function. For example, previous studies have shown that insulin increases the release of leptin from adipose tissue [START_REF] Gettys | The β3-adrenergic receptor inhibits insulin-stimulated leptin secretion from isolated rat adipocytes[END_REF] while cyclic AMP inhibits both leptin expression and release [17; 98]. In addition to promoting triglyceride accumulation, the higher insulin levels would favor increased leptin expression and release from adipose tissue during this period.

Although beyond the scope of this minireview, it is also likely that other components of the adipocyte endocrine repertoire that are regulated by insulin and cyclic AMP would be similarly affected.

Lastly, leptin regulates SNS outflow to adipose tissue, which acts through cyclic AMP to mobilize triglyceride and transcriptionally remodel adipose tissue. However, the increased serum insulin levels would antagonize cyclic AMP-dependent signaling, effectively limiting the efficacy of leptin-dependent SNS input. Viewed collectively, the promotion of hepatic IR of sufficient magnitude to increase circulating insulin, but not induce IR in peripheral tissues, may be a critical characteristic of the response to the 45 kcal% version of the HF diet which promotes rapid fat deposition and the subsequent development of leptin resistance. It seems clear that detailed longitudinal studies at much earlier time points will be required to resolve how and where the signaling defects develop, where they interact, and how they impact the expansion of adipose tissue mass.

VII. Conclusions

It

  is well accepted that postweaning consumption of diets high in saturated fat produces obesity and metabolic dysfunction. The severity and developmental progression of this syndrome is dependent upon the amount of dietary fat (45-60 kcal%), the strain of mouse being studied, and the A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT age at which exposure to the diet is initiated. Our theoretical model (Figure 1) is based on postweaning consumption of a 45 kcal% high fat diet by C57BL/6J mice, and proposes that the development of insulin resistance is progressive and tissue specific, with high-fat diets leading to hepatic insulin resistance prior to detectable changes in insulin sensitivity in peripheral tissues. The resulting increase in circulating insulin, in conjunction with retention of peripheral insulin sensitivity, produces an expansion of adipose mass and a concomitant increase in circulating leptin. This increase in leptin, though perhaps initially acting to limit body adiposity, leads to hypothalamic leptin resistance and a further dysregulation of energy balance and glucose homeostasis. With continued exposure to a high fat diet, the liver becomes progressively less responsive to insulin, vagal inhibition of hepatic glucose production is diminished, and the resulting oversupply of lipids to mitochondria of peripheral tissues produces a mismatch between TCA cycle flux and fat oxidation (Figure 1, Panel C). The result is incomplete fatty acid oxidation, accumulation of lipotoxic short chain fatty acylcarnitines, and peripheral insulin resistance. Adipose tissue in particular becomes resistant to inhibition of leptin release by SNS input, resulting in chronic hyperleptinemia and leptin resistance. Lastly, we recognize the possibility that excess dietary fat may also more directly induce hypothalamic leptin (and insulin) resistance, with this effect serving to exacerbate the initial deterioration peripheral insulin sensitivity and whole body energy homeostasis. A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

Figure 1 -

 1 Figure 1 -Interactions between insulin and leptin signaling in mice reared on low fat diets (Panel A),

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