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Choosing Lunch: The Role of Selective Autophagy Adaptor Proteins Choosing Lunch: The Role of Selective Autophagy Adaptor Proteins

Autophagy (macroautophagy) is a lysosome-dependent catabolic pathway that degrades damaged organelles, protein aggregates, microorganisms, and other cytoplasmic components. Autophagy was previously considered to be nonselective; however, studies have increasingly established that autophagy-mediated degradation is highly regulated. Selective autophagy regulates plenty of specific cellular components through specialized molecules termed autophagy receptors, which include p62, NBR1, NDP52, optineurin, and VCP among others. Autophagy receptors recognize ubiquiti- nated cargo and interact with the LC3/GABARAP/Gate16 protein on the membrane of nascent phagophore. In this review, we summarize the advances in the molecular mechanisms of selective autophagy adaptor proteins. Kimonis Yao TP, et al. VCP/p97 is essential for maturation of ubiquitin-containing autophagosomes this function is

Introduction
The various functions of eukaryotic organisms depend largely on the existence of highly efficient regulation mechanisms. Each physiologic activity involves the production of several molecules whose half-life must be controlled by degradation system to maintain homeostasis. Up to the present time, two systems of degradation molecules or organelles are known: (a) ubiquitin-proteasome system (UPS) and (b) autophagy, a lysosome-dependent degradation system. The precise mechanisms to lead the substrate to UPS or autophagy are not understood completely. However, it is known that ubiquitin is a key protein to regulate the substrate recognition through the conjugation of a single ubiquitin monomer (monoubiquitination) or sequential conjugation of several ubiquitin moieties (polyubiquitination). The conjugation of four ubiquitin monomers is sufficient signal to allow the ubiquitylated target protein to be recognized by UPS [1]. The specificity in the UPS is generated by the ability of ubiquitin to form eight different chain linkages on itself, through its seven lysine residues (K6, K11, K27, K29, K33, K48, and K63). K48 ubiquitin chain is the most well studied and was originally identified as the signal to target proteins to proteasomal degradation [2]. K11 and K63 ubiquitin chains are more related to signal for nonproteolytic functions as DNA repair and cell signaling, but a recent study shows that heterotypic K11/K48-polyUb chains bind to the proteasome and facilitate the degradation of cyclin B1 [3]. Whereas UPS is the major degradation pathway for short-lived and regulatory proteins, autophagy is more linked with the elimination of long-lived proteins and organelles. The selectivity of autophagy degradation is conferred by K63 ubiquitin chains [4,5] (Figure 1). Autophagy was first described as a nonselective bulk degradation system, and now the accumulated evidence indicates that autophagy can be highly selective. Nonselective autophagy is triggered as a response to starvation and implies the random formation of the autophagosomes with the subsequent capture of any organelle or molecule near the autophagosome. In contrast, the selective autophagy is involved in the recruitment of different adaptor proteins that interact with Atg proteins and target organelles or molecules to be degraded [6,7]. It is possible to distinguish Figure 1. Schematic representation of selective autophagy. The two degradation pathways are shown. Mitochondria, misfolded proteins, and microorganisms are ubiquitinated and selected to proteasome system or autophagy. Selective autophagy involves the participation of adaptor proteins as p62, NDP52, optineurin, NBR1, and VCP, as bridges to cargo and nascent phagophore. various types of selective autophagy, depending on the cargo that is captured and degraded: lipid droplets (lipophagy), mitochondria (mitophagy), ER (reticulophagy), pathogens (xenophagy), and aggregation-prone proteins (aggrephagy) [8], among others. Nevertheless, the precise mechanism of cargo recognition remains unclear; the molecular characterization of autophagy receptors, initially SQSTM1/p62 and NBR1, has revealed that ubiquitination is involved in substrate selectivity. Most autophagy receptors have a ubiquitin-binding domain (UBD) and LC3-interacting region (LIR). The UBD domains attach to target molecules or organelles, and LIR domain interacts with LC3/GABARAP/Gate16 protein in autophagy to facilitate autophagosome formation, transport, and/or maturation [8]. The core of LIR consists of D/E, D/E, D/E, F/W/Y, X, X, L/I/V, and D; the phosphorylation of this domain enhances the affinity with LC3/GABARAP/Gate16 protein [7]. In this chapter, we discuss the recent knowledge on autophagy receptors and their role in selective autophagy.

SQSTM1/p62
SQSTM1/p62 (referred to hereafter as p62) was initially identified as a phosphotyrosineindependent binding of a 62 kDa (gave p62 its name) to the Src homology 2 (SH2) domain [9]. Subsequently, the term sequestosome-1 (SQSTM1) was assigned for its capacity to sequester polyubiquitinated protein to cytoplasmic storage before its degradation by the proteasome, protecting the cytosol from the toxic effect of misfolded proteins [10]. Currently, p62 has been considered a protein with pleiotropic activities, derived from their multiple domains, which interact with several molecules involved in the cellular death, oxidative stress, inflammatory response, and recognition of molecules to be degraded by UPS or autophagy [10,11]. Recent studies have reported that mTOR activation depends on p62 as a key regulator of the nutrientsensing pathway [12].  p62 is a multifunctional protein of 440-amino acids that is conserved in metazoans but not in plants and fungi [13]. Refractory to Sigma P (Ref(2)P) is a homologue protein of mammalian p62 in Drosophila melanogaster, that regulates protein aggregation in adult brain [14]. p62 has six functional domains: the N-terminal Phox/Bem1 domain (PB1, 21-103 aa), a ZZ-type zing finger domain (ZnF, 128-163 aa), TRAF6-binding domain (TB, 225-250 aa), a short LC3interacting region (LIR, 321-345 aa), KEAP1-interacting region motifs (KIR, 346-359), and ubiquitin-associated domain (UBA, 386-440 aa) which is localized in C-terminal end ( Figure  2) [11].
PB1 domains, ZZ zinc finger, KIR, and UBA can bind several proteins to participate in inflammatory responses and receptor-mediated signal transduction. PB1 has been associated with atypical protein kinase C (aPKC) to activate NF-kB signaling pathway. The ZZ zinc finger domain is responsible for binding the receptor interacting protein (RIP) to regulate the inflammatory response. The KIR region of this scaffold protein is a regulator for Nrf2, and its activation induces transcription of oxidative stress response genes [11,15].
p62 provides a link between the degradation of ubiquitin cargo by UPS or autophagy through the UBA domain. The PB1, LIR, and UBA domains are implied in the degradation of ubiquitinated cargo by selective autophagy. UBA domain is responsible for noncovalent binding with polyubiquitinated cargos, through serine 403 phosphorylation by casein kinase 2 (CK2), which increases the affinity for ubiquitinated chains [16,17]. PB1 domain is involved in self-and heteroligomerization with NBR1 (another receptor of selective autophagy) [18][19][20], and LIR region is important to LC3 interaction. It has been hypothesized that the activity of p62, in selective autophagy, requires a sequential interaction. Initially, there must be an interaction of p62 with ubiquitin proteins, and then aggregation of complex protein oligomerization to itself or with NBR1, and these aggregates are finally degraded by autolysosomes [7,21,22]. This order is altered in defects of autophagy, where firstly accumulations of p62 proteins are present and later are ubiquitinated [23,24].
Furthermore, p62 is an autophagy receptor that can bind proteins to be degraded by selective autophagy as aggregates of misfolded proteins, damaged mitochondria, peroxisomes, and intracellular bacteria, which are ubiquitinated and targeted for clearance by autophagy [7,25]. Several works have evidenced that p62 has a critical role in the normal functioning of mitochondria. p62 is localized to mitochondria under physiological conditions and plays an important role in mitochondrial morphology, genome integrity, and mitochondrial import of transcription factors. When p62 is deleted, it leads to mitochondrial fragmentation and mitochondrial dysfunction [26]. The role of p62 as adaptor receptor in mitophagy is currently debated. Geisler and colleagues reported that p62 with PINK1 y Parkin molecules has a key role in the sequential mitophagy process. p62 colocalized with Parkin on clustered mitochondria after the induction of mitophagy by carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment, and the silence of p62 by siRNA resulted in a significant loss of mitochondrial clearance [27,28]. In contrast, Narendra and colleagues mentioned that p62 is not indispensable in the mitophagy; in HeLa cells with siRNA directed against p62, no difference was found in lacking mitochondria after the induction of mitophagy but is important in clustering of depolarized mitochondria [29,30]. Similarly, in pexophagy, the role of p62 is not clear. p62 has been involved in the clustering of peroxisomes that were labeled with ubiquitin to selective degradation [31], but recent work suggests that p62 is responsible for clustering, and only NBR1 is essential for peroxisome degradation by autophagy [32].
Adaptor p62 protein has an important role in the xenophagy, which is responsible for restriction of the replication of several intracellular microorganisms. The role of p62 has been more explored in infections by intracellular bacteria. Bacteria such as Shigella flexneri, a nonmotile actA mutant of Listeria monocytogenes [33], Salmonella enterica serotype Typhimurium [34], and Mycobacterium tuberculosis [25,35] are targeted selectively through p62 recruitment to deliver into nascent LC3-positive isolation membranes for autophagosomal degradation. Interestingly, p62 has an additional role in antibacterial effect against M. tuberculosis, through the delivery of cytosolic proteins to M. tuberculosis containing-autolysosomes, where they are processed to convert into new antimicrobial peptides [36]. In viral infections, it has been reported that p62 plays a role in the clearance of viral proteins. Orvedahl and colleagues demonstrate that Sindbis virus capsid protein can interact with p62 in an ubiquitin-independent pathway, suggesting that clearance of viral proteins by autophagy requires p62 and other molecules tag different from ubiquitin [37]. There is little evidence about the role of p62 in infections by parasites. In the infection of Toxoplasma gondii, it has been observed that p62 and ubiquitin were recruited to T. gondii parasitophorous vacuoles when infected cells were stimulated by INF-γ playing an important role in the antigen presentation to activate specific CD8+T cells [38].
The high relevance of p62 as a signaling hub implies their efficient regulation. When p62 is disregulated or dysfunctional, there are multiple consequences. Several studies have implicated p62 aggregates in cancer, inflammation, neurodegenerative disease, liver disease, and aging [10,13,20,39].

NBR1
Neighbor of BRCA1 gene 1 (NBR1) is another cargo receptor that is selectively degraded by autophagy. NBR1 was originally cloned as a candidate gene for the ovarian cancer antigen CA125 [40]. Given the similarity and interaction with p62, NBR1 has been studied in cell signaling and differentiation [41]. In 2009, Kirkin and colleagues have shown that NBR1 was involved in autophagic degradation of ubiquitinated targets [42].
NBR1 was recognized as a direct binding partner of the autophagosome-specific ATG8/LC3/ GABARAP modifiers both in vitro and in vivo. NBR1 has similar domain architecture as p62 and shares several key features with p62 though differs in sequence and size. Both proteins share a very similar overall domain architecture, consisting of an N-terminal PB1(residues 5-85) domain, a ZZ-like zinc finger domain (residues 215-259), a two-domain light-chain-3interacting regions LRS1 (residues 540-636) and LRS2 (residues 727-738, the LRS2 does not have the core consensus motif W/YXXL/I, most likely representing a novel type of LC3interacting sequence), and a C-terminal ubiquitin-associated (UBA) domain (Figure 2) [43].
NBR1 binds strongly to ubiquitin via its UBA domain with a bias toward the K63-linked polyUb chains [43]. NBR1 undergoes dimerization via the coiled-coil domain. NBR1 can directly bind to p62, and together they act as cargo receptors for autophagic degradation of polyubiquitylated aggregates and peroxisomes [43][44][45]. In the absence of p62, NBR1 interacts with misfolded and ubiquitinated proteins for degradation by autophagy [42]. Moreover, it is known that NBR1 promotes cell differentiation, may act as a tumor suppressor, and is also involved in bone remodeling [46,47]. NBR1 also is involved in protein misfolding disorders such as body myositis sporadic inclusion, and autophagic degradation may have a role in the pathology [48].

NDP52
The nuclear dot protein 52 kD (NDP52) also named as calcium binding and coiled-coil domain 2 (CALCOCO2) is a 446-amino acid protein. Discovered in 1995, it was first erroneously found as part of the nuclear domains (ND10) called Kr bodies or PLM-containing oncogenic domains (POD), consisting of protein aggregates detected as dots (approximately 10) by autoimmune sera or monoclonal antibodies. By 1997, Sternsdorf and colleagues found by using polyclonal sera anti-NDP52 that the protein localization is restricted to the cytoplasm but not ND10 and confirmed an increase of NDP52 transcripts when cells were treated with IFN-γ [49].
NDP52 has a predicted molecular mass of 52 kD and exhibits an N-terminal skeletal muscle, and kidney-enriched inositol phosphatase carboxyl homology (SKICH) domain (aa 1-127), a central coil-coiled domain with zipper leucine motifs (aa 140-420) and the C-terminus presents homology with Lin11, Isl-1, and Mec-3 (LIM) domains containing two zinc finger arrangements involved in protein-protein interactions as ubiquitin (Figure 2) (aa 421-446) [50][51][52]. There are two paralogs existing, CoCoA (also known as Calcoco1) and Tax1BP1 (also known TXBP151). The CoCoA paralog comprises SKICH domains and LIM domains as NDP52, but it lacks an ubiquitin-binding domain. Therefore, it did not decorate bacteria when they escaped to the cytosol [53].
First studies linking NDP52 to a physiological process showed that after infection with S. enterica, the ubiquitin-coated bacteria in the cytosol are recognized by NDP52, which acts as a receptor. Then, NDP52 interacts with the adaptor proteins, Nap1 or Sintbad (also named TBKBP), and leads to the recruitment of TANK-binding kinase 1 (TBK1), which results in the control of bacterial growth [52]. The authors also found that NDP52 recruits and binds ATG8/ LC3, an autophagosomal marker, and the knockdown of NDP52 impairs the autophagy of Salmonella [52,54]. The same effect was observed with Streptococcus pyogenes-infected cells but not with S. flexneri. They conclude that NDP52 is a receptor that recognizes ubiquitin-coated bacteria and binds ATG8/LC3 leading to the control of bacterial growth by autophagy. Further studies demonstrated that NDP52 has a LC3-interacting region (LIR) domain and the sites of interaction with Nap1 or Sintbad are located at the SKICH domain (Figure 2) [55]. Later, Muhlinen and colleagues demonstrated that NDP52 binds all human ATG8/LC3 orthologs (LC3A, LC3B, LC3C, GABARAP, GABARAPL1, GABARAPL2), but only LC3C performs an antibacterial function when binds NDP52 through the LC3C-interacting region (CLIR) [56].
Other studies demonstrated that S. flexneri is also targeted by NDP52/P62 to autophagy pathway dependent upon septin and actin [57]. Additional to the ubiquitin-dependent pathway needed for the recruitment of NDP52, a carbohydrate-dependent galectin-8 pathway also mediates NDP52 recruitment to invading bacteria at early stages of infection, unlike the ubiquitin-dependent pathway which plays a major role at later points [58,59].
As part of the autophagosome maturation, it was found that NDP52 interacts with myosin VI via RRL motif, and such interaction recruits myosin VI to deliver endosomal membranes to the nascent autophagosome [60,61]. This function of NDP52 is independent of its function in xenophagy and involves a different binding domain [62].
Recently, it has been demonstrated that NDP52 also plays a role in regulation. The Toll-like receptor (TLR) signaling serves as, an example, the selective autophagic degradation of Toll/ interleukin-1 receptor homology domain-containing adaptor-inducing interferon (TRIF), and TRAF6 is mediated by this receptor. The mechanism involves the polyubiquitination of NDP52 by TRAF6 to acquire the ability to form aggregates of polyubiquitinated TRAF6 [63,64]. The regulation of the miRNA activity is another example; recently, it has been discovered that the miRNA-processing enzyme DICER and the miRNA effector AGO2 are a target by NDP52 for their degradation via autophagy [65]. Another target of NDP52 includes the RNA retrotransposon, and the degradation of this RNA via autophagy helps to maintain the stability of the genome [66]. More recently, Heo and colleagues found that the PINK1-PARKIN mitochondrial ubiquitination pathway promotes mitophagy by recruiting TBK1 kinase which binds to NDP52 and other autophagy receptors to induce autophagy of mitochondria [67,68].
Some studies with viruses have related autophagy to anti-or pro-viral roles; NDP52 has been involved in promoting viral replication of chikungunya virus (CHIKV) when interacts with the nonstructural protein nsP2 in infected human cells [69].
Although autophagy has a protective role against some intracellular bacteria, some studies indicate that NDP52 has a relation with Crohn's disease, and Ellinghaus and colleagues found an association between the disease and a missense mutation in affected individuals [70,71]. Additionally, NDP52 has been involved in Alzheimer's disease where it has a protective role in facilitating the clearance of phosphorylated tau [72,73].

Optineurin
Optineurin was first described by Li and colleagues [74]. They were looking for proteins that interact with the early region 3 (E3) 14.7 K protein in the yeast two-hybrid system. E3 14.7 K is synthesized by E3 in group C of adenovirus and is an inhibitor of NF-kB cytolysis. They found a protein with the ability to interact with E3 14.7K and named as FIP-2 (for 14.7K interacting protein), and FIP-2 interacts with E3 14.7K in the cytoplasm and caused redistribution of the protein. Also, FIP-2 reversed the protective effect of E3 14.7K on cell death induced by TNF-α. After, Schwamborn and colleagues described that FIP-2 had a strong homology to NF-kB essential modulator (NEMO) and named as NEMO-related protein (NRP). They found that NRP was associated with Golgi apparatus and is de novo expressed by interferon and TNF-α [75]. Rezaie and colleagues coined the name optineurin (optic neuropathy-inducing protein) after discovering that this molecule was associated with diseases such as normal tension glaucoma and a subtype of primary open-angle glaucoma [76].
Optineurin is a 67 KDa intracellular protein found in different tissues [77], and optineurin gene encodes an 884-amino acid protein and contains three noncoding exons and 13 exons that code for a 577-amino acid protein [78]. The mRNA can be found in 3 isoforms as a result of alternative splicing [74]. Optineurin has been described in different tissues such as spleen, kidney, skeletal muscle, brain, heart, lung, pancreas, and eyes of various species: human, mouse, and chicken [74,78,79]. The UPS is a very important pathway to recycle optineurin, but in situations where optineurin is upregulated, the UPS is compromised and autophagy is induced to control the optineurin levels [80].
Optineurin has different domains through which it can interact with different proteins. It contains putative domains such as C-terminal zinc finger, leucine zipper domain [74], a LIR domain [81], a NEMO-like domain [75], UBD domain [82], and various coiled-coil motifs [83].
In Figure 2, optineurin is shown and compared with other adaptor proteins involved in selective autophagy.
Optineurin can participate in different biological activities because it has multiple domains, which mediate the interaction with other proteins. For example, optineurin can interact with Rab8 [83][84][85], transferrin receptor [86], serine/threonine kinase receptor-interacting protein 1 (RIP) [87], ubiquitin [88], and Myosin VI [61,84], among others. The interaction of optineurin with myosin VI [84,89] is mediated by the UBD domain, and this interaction is important in the fusion of secretory vesicles with the plasma membrane [90]. Also, it has been described that macrophages from patients with Crohn's disease where optineurin was under-expressed fail to secrete pro-inflammatory cytokines [91], which suggest that optineurin can also be involved in the vesicular transport of the autophagosomes.
As it was mentioned, the importance of optineurin in selective autophagy relies on their UBD and LIR domains, through their interaction with specific cargo and the autophagy machinery respectively. However, it has been reported that optineurin can recognize a target like a superoxide dismutase 1 and huntingtin protein by an ubiquitin-independent pathway and degrade protein aggregates through autophagy [92], and this recognition was related to the C-terminal coiled-coil domain of optineurin.
LIR domain mediates the interaction of optineurin with autophagy machinery. The phosphorylation of serine 177 on LIR domain by TANK-binding kinase 1 (TBK1) increases the affinity of optineurin to LC3/GABARAP proteins [81,93]. After the dominant phosphorylation at serine 177, optineurin forms a strand with the beta-strand 2 of LC3B and phenylalanine 178 and isoleucine 181 are inserted into a hydrophobic pocket on the LC3B [93]. On the other hand, the isomers of LC3 and the proteins of the GABARAP family interact with the machinery involved in autophagosome elongation, which is recruited to LC3/GABARAP-optineurin. Then, autophagy-related protein ATG4 is recruited to cleavage at the C-terminal of LC3/ GABARAP and exhibits phenylalanine and glycine amino acids, which participate in the conjugation of LC3/GABARAP with phosphatidylethanolamine (PE) [94] to complete autophagosome formation finally.
Mitochondria are important and dynamic organelles, depending on energetic requirements of the cells, mitochondria can undergo cycles of fusion and fission [95]. When mitochondria are damaged, they suffer an increase in the rate of fission, and this results in their fragmentation. Damaged and fragmented mitochondria are removed via mitophagy [28,96]. The role of optineurin in mitophagy has been recently studied. Mitophagy requires the interaction with other proteins. First, damaged mitochondria are marked with ubiquitin by Parkin and PINK1. Both proteins act in the ubiquitination of Mitofusin 1 and Mitofusin 2 when mitochondrial depolarization was induced by carbonyl cyanide-m-chlorophenylhydrazone (CCCP) treatment [97]. When mitochondria become depolarized, PINK1 accumulates on the mitochondrial outer membrane and phosphorylates to Mitofusin 2, this allows their interaction with inactive Parkin, and the subsequent activation of Parkin by PINK1 activates the Parkin ubiquitin ligase activity [98]. This ubiquitination allows the interaction of optineurin with ubiquitinated mitochondria through its UBD domain, and this recognition is similar to p62 and NIX [28]. After this initial recognition, optineurin recruits autophagy machinery around damaged mitochondria to capture it into the autophagosome [96].
Xenophagy has been considered as an innate immune response against intracellular infections. Xenophagy guided by optineurin has been poorly described, so it represents an interesting and wide field of study. It has been described that xenophagy mediated by optineurin participates in the intracellular control of S. enterica. For this activity, optineurin requires UBD and LIR domains. It has been reported than when UBD domain was mutated, optineurin failed to colocalize with S. enterica and when LIR domain was mutated, optineurin colocalized with S. enterica but not with LC3. Also, TBK1 activity was necessary for the xenophagy mediated by optineurin [81]. Tumbarello and colleagues found that optineurin, TAX1BP1, and NDP52 are important in xenophagy response against Salmonella typhimurium. Also, they found that Myosin VI is necessary to restrict the replication of S. typhimurium, highlighting the role of Myosin VI in the vesicular transport of autophagosomes containing S. typhimurium to lysosomes [99].
Due to the role of optineurin in the capture of unnecessary or damaged organelles, the lack or the deficiency of optineurin has been associated with different pathologies such as amyotrophic lateral sclerosis [100], Paget's disease of bone [101], normal tension glaucoma, and primary open-angle glaucoma [102].

VCP/p97
Valosin-containing protein (VCP/97)-also called Cdc48p in yeast, p97 in Xenopus, CDC-48 in  2). VCP proteins form a barrel-like structure that comprises two ring-shaped layers made of D1 and D2 AAA modules [103]. The diversity of cellular functions and the activity of VCP/p97 are mediated by their interaction with a large number of protein cofactors. p97 forms core complexes with the major cofactors, which include the proteins ubiquitin-X (UBX) domain, the Ufd1 (ubiquitin fusion degradation 1)-Npl4 (nuclear protein localization homolog 4) heterodimer, and p47 [104]. Several works have established p97 as a principal element in emerging functions of the UPS as was described in a review by Meyer et al. [104]. Nevertheless, p97 lacks a LIR domain; recent reports now link to p97 with the autophagy. The first findings of the involvement of p97 with autophagy were reported in studies of the multisystem degenerative disorder characterized by inclusion body myopathy, frontotemporal dementia, and Paget's disease of bone (also known as IBMPFD) [105] which is associated with VCP mutations. This disorder is characterized by the extensive accumulation of ubiquitin conjugates in affected tissues, and in IBMPFD patients the VCP mutations cause no damage in ubiquitin-dependent degradation by the proteasome, but do impair maturation of ubiquitincontaining autophagosomes. Further, the myoblasts derived from IBMPFD patients showed accumulation of LAMP-1, LAMP-2, and LC3-II-positive vacuoles indicating that VCP/p97 is essential to autophagosome maturation [106]. Additional work has provided evidence about the role of Cdc48/p97 in the regulation of autophagosome biogenesis. In Saccharomyces cerevisiae, it has been demonstrated that the participation of Cdc48/p77 in autophagy is mediated by direct interaction of Shp1/Ubx1 cofactor with Atg8 PE-conjugated form [107].
Recently, it has been recognized the role of VCP/p97 in mitochondrial maintenance. On the one hand, VCP/p97 was accepted as part of outer mitochondrial membrane-associated degradation (OMMAD), functioning as retrotranslocase of ubiquitinated mitochondrial proteins for degradation by UPS [108]. On the other hand, Tanaka and colleges showed that p97 and proteasome activity are required to mitophagy mediated by Parkin protein [109]. The role of VCP/p97 in xenophagy remains unexplored, which opens a new window to investigate.

Conclusion
Currently, autophagy is an attractive area of investigation. It has been recognized the participation of autophagy in homeostatic cellular functions, such as clearance of damaged organelles, misfolded proteins, and microorganism, among others. Scientists have focused on describing the molecular mechanisms responsible for the autophagy. Now, we know that autophagy, far from beginning a random pathway, is a mechanism that elegantly regulates and is highly orchestrated by several proteins. Some proteins have been identified as bridges between the cargo and nascent phagophore, and recently studies are in process to know how these proteins are working and how to interact with the complex autophagy machinery. Several of these proteins share some structural characteristics, such as LIR domain, which allow the direct interaction with LC3 protein. However, recently, studies have identified new proteins that participate in selective autophagy but lack LIR domain, for example, VCP/p97 and Alfy. The studies to know the precise mechanisms of interaction of these proteins are in process. The understanding of the molecular mechanism that governs the autophagy represents an interesting field because many of these molecules could be manipulated to recover the cellular homeostasis in several pathologies, where autophagy is involved.


s2fieldsofstudy: Biology Medicine

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The Interplay between the Renin Angiotensin System and Pacing Postconditioning Induced Cardiac Protection

Background Accumulating evidence suggests a cardioprotective role of pacing postconditioning (PPC) maneuvers in animal models and more recently in humans. The procedure however remains to be optimized and its interaction with physiological systems remains to be further explored. The renin angiotensin system (RAS) plays a dual role in ischemia/reperfusion (I/R) injury. The interaction between RAS and PPC induced cardiac protection is however not clearly understood. We have recently demonstrated that angiotensin (1–7) via Mas receptor played a significant role in PPC mediated cardiac protection against I/R injury. Objective The objective of this study was to investigate the role of angiotensin converting enzyme (ACE)—chymase—angiotensin II (Ang II)—angiotensin receptor 1 (AT1) axes of RAS in PPC mediated cardiac protection. Methods Isolated rat hearts were subjected to I/R (control) or PPC in the presence or absence of Ang II, chymostatin (inhibitor of locally produced Ang II), ACE blocker (captopril) or AT1 antagonist (irbesartan). Hemodynamics data was computed digitally and infarct size was determined histologically using TTC staining and biochemically by measuring creatine kinase (CK) and lactate dehydrogenase levels. Results Cardiac hemodynamics were significantly (P<0.001) improved and infarct size and cardiac enzymes were significantly (P<0.001) reduced in hearts subjected to PPC relative to hearts subjected to I/R injury. Exogenous administration of Ang II did not affect I/R injury or PPC mediated protection. Nonetheless inhibition of endogenously synthesized Ang II protected against I/R induced cardiac damage yet did not block or augment the protective effects of PPC. The administration of AT1 antagonist did not alleviate I/R induced damage. Interestingly it abrogated PPC induced cardiac protection in isolated rat hearts. Finally, PPC induced protection and blockade of locally produced Ang II involved enhanced activation of ERK1/2 and Akt components of the reperfusion injury salvage kinase (RISK) pathway. Conclusions This study demonstrate a novel role of endogenously produced Ang II in mediating I/R injury and highlights the significance of AT1 signaling in PPC mediated cardiac protection in isolated rodents hearts ex vivo. The interaction between Ang II-AT1 and PPC appears to involve alterations in the activation state of ERK1/2 and Akt components of the RISK pathway.

Introduction
Cardiovascular diseases are major health concerns worldwide and coronary heart disease (CHD) continues to be a leading cause of death [1]. Intermittent interruption of coronary flow in the early phases of reperfusion, postconditioning, has proven to be cardioprotective [2]. Recently however well planned postconditioning clinical studies resulted in disappointing negative outcomes [3][4][5][6] thus necessitating the requirement of alternative procedures that can successfully be translated to the clinic [7]. Pacing post conditioning (PPC) is a modification of classical ischemic postconditioning that involves changing the stretch pattern of the heart by intermittent dyssynchrony of the left ventricle (LV) contraction at a remote site away from the affected blood vessel [8]. We have previously demonstrated a pronounced reduction in the infarct size and a significant improvement in cardiac hemodynamics in hearts subjected to PPC [9,10]. Recent introduction of PPC to the clinic demonstrated promising results in STsegment elevation myocardial infarction (MI) patients [11]. When combined with percutaneous coronary intervention, PPC consisting of 10 episodes of 30 sec right ventricle pacing resulted in a 25% reduction in infarct size [11]. Despite the observed cardioprotection PPC however, resulted in an increased incidence of ventricular fibrillation and paroxysmal atrial fibrillation [11]. This could be due to prolonged pacing as continuous prolonged pacing was proven to be detrimental to the heart [12]. PPC thus appears to be a potentially attractive target. However optimization of PPC maneuver and in depth understanding of its interaction with potentially relevant physiological mediators are challenges that remain to be addressed for an appropriate application of the technique in the clinic.
The renin-angiotensin system (RAS) is a master regulator of cardiovascular physiology and remolding [13]. Key effector peptides of the RAS include angiotensin II (Ang II) and angiotensin (1-7) acting via angiotensin receptors-1 or -2 (AT1 or AT2) and Mas receptors, respectively [14]. While Ang II generally demonstrated to have detrimental effects on the development and progression of cardiovascular disease [15][16][17], Ang (1-7) was shown to be protective [18]. The exact role of RAS in PPC mediated protection is however not well defined. We have recently demonstrated the involvement of Ang (1-7)-Mas receptor axes in the cardioprotective effects of PPC in isolated rodent hearts [18]. In this study however we were set to determine the role of the other arm of the RAS, specifically Ang II, in PPC mediated cardiac protection. Ang II is a biologically active octapeptide that can be produced systemically by the action of angiotensin converting enzyme (ACE) on circulating angiotensinogen [13] or locally via ACE dependent [19] and independent enzymes including chymase, cathapsin G and Kallikrein [20,21]. Evidence from clinical studies indicate enhanced chymase activity relative to other Ang II forming enzymes including ACE and cathapsin G in the myocardium 14-21 days post myocardial infarction (MI) suggesting the importance of chymase in post ischemic damage to cardiomyocytes [22]. The role of systemically produced or endogenously synthesized Ang II in PPC mediated protection is however not clearly understood. We hypothesize that exogenously infused and/or locally produced Ang II, acting via AT1, to negatively impact PPC mediated cardiac protection. The objective of this study was therefore to investigate the involvement of ACEchymase-Ang II-AT1 axes in PPC mediated cardiac protection and map potential downstream signaling pathways.

Ethics statement
The study was approved by Health Science Center, Kuwait University Ethics Committee for animal use. The study was conducted according to the laboratory's animal care guidelines at Kuwait University, Kuwait in accordance with the international standards of animal care. Rats were maintained at 22°C on 12-hr light/dark cycle (7 am-7 pm) and water and food were available ad libitum. Rats were anesthetized with intraperitoneal injection of sodium pentobarbital (60 mg/kg) and heparin (1000 U/kg) was administered through the femoral vein, then were sacrificed by cervical dislocation.

Animals and Instrumentation
Hearts isolated from male Wistar rats weighing between 270-300 g were used for this study. Heart cannulation and perfusion were performed as described previously [27]. Briefly, isolated hearts were perfused retrogradely with freshly prepared Krebs-Hensleit buffer, pH 7.35 to 7.45 at 37.0 ± 0.5°C and gassed with CO 2 (5%) and O 2 (95%). Pacing electrodes were placed on RA appendage to keep the heart beating at physiological rat heart rate. Regional ischemia was induced by occluding left anterior descending (LAD) coronary artery for 30 min. Preload was kept constant at 6 mmHg under basal controlled conditions and perfusion pressure (PP) at 50 mmHg throughout the experimental procedure in all protocols. The perfusion pressure was measured downstream from a branch of the aortic cannula using a "statham pressure transducer" (P23 Db). Constant PP was ensured electronically by means of the perfusion assembly ("Module PPCM type 671 (Hugo Sachs Elektronik-Harvard ApparatusGmbH, Germany")), an effective system for the accurate adjustment of PP between 5 mmHg to 150 mmHg with ± 1 mmHg accuracy level.

Study Protocol and Study Groups
Hearts were subdivided into 10 groups (n = 8 per group). Hearts were subjected to ischemia followed by reperfusion (control) or ischemia followed by LV pacing (PPC) in the presence or absence of target specific drug (Fig 1). Ischemia (30 min) was produced by LAD coronary artery occlusion. The LAD coronary artery was encircled by a snare at approximately 0.5 cm below the atrioventricular groove, and a small rigid plastic tube was positioned between the heart and the snare to ensure complete occlusion of the coronary artery. Hearts were then reperfused for 30 min. In hearts subjected to PPC, one pacing electrode was fixed to the posterior basal LV wall and connected to a pacemaker set to the required pacing frequency. PPC involved three alternating RA and LV pacing episodes, 30 s each. The on and off cycles of pacing were selected based a previous study by our group [10] and were confirmed by a pilot study (data not shown).

Data Collection and Processing
LV function was evaluated by the assessment of LV end diastolic pressure (LVEDP) and LV developed pressure (DPmax) while coronary-vascular dynamics were evaluated by the assessment of the coronary flow (CF) and coronary vascular resistance (CVR). Cardiovascular functions were measured as previously described [27]. Briefly, a water-filled latex balloon was placed and secured in LV cavity. The balloon was attached to a pressure transducer and a "DC-Bridge amplifier (DC-BA)" with a pressure module (DC-BA type 660, Hugo-Sachs Electronik, Germany) and interfaced to a personal computer for on-line monitoring of DPmax. LV developed pressure was derived from online acquisition of LV end systolic pressure (LVESP) using Max-Min module (Number MMM type 668, Hugo Sachs Elektronik-Harvard Appara-tusGmbH, Germany) which has the ability to convert the output from DC bridge amplifier to DPmax by subtracting LVEDP from the LVESP.
Coronary flow was continuously measured using an electromagnetic flow probe attached to the inflow of the aortic cannula interfaced to a personal computer. The continuous monitoring of the CF in ml/min was digitally monitored using software developed by Hugo-Sachs (Hugo-Sachs Electronik, Germany) specifically for this purpose and was manually verified by timed collection of coronary effluent. The CVR and hemodynamics data were determined every 10 sec using an on-line data acquisition program (Isoheart software V 1.524-S, Hugo-Sachs Electronik, Germany). By the end of each experiments, hearts were snap frozen in liquid nitrogen then stored in -80°C for further analysis.

Assessment of cardiac damage by TTC staining and cardiac enzyme release
Hearts (n = 4 per group) were collected after 30 min of reperfusion and stored overnight at -20°C. The next day the hearts were sliced to 4-5 pieces from apex to base. The slices were then incubated in 1% 2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) solution in isotonic (pH 7.4) phosphate buffer and then fixed in 4% formaldehyde. The red and pale non stained areas of every slice were indicated manually on the image. The percentage of the infarct size of the LV was calculated for every heart. Quantification of the LV and infarct size was done using ImageJ (Image J, Wayne Rasb and National Institute of Health, USA). Cardiomyocytes injury was evaluated by measuring creatine kinase (CK) and lactate dehydrogenase release in the coronary effluent during the reperfusion period as described previously by Ferrera et al. [28].

Immunoblotting
Left ventricles (n = 4 per group) were homogenized in ice-cold lysis buffer, and the homogenate was centrifuged at 4000 rpm for 10 min. The supernatant was collected, protein content was measured and the samples were aliquoted and stored at -80°C for further analysis. The expression of proteins in question was done as described previously [29]. Equal loading was checked by stripping and reprobing the membrane with Actin antibodies. The levels of total ERK1/2, p38 MAPK and AKT and phosphorylated ERK1/2 (Thr202/Tyr204), phosphorylated p38 MAPK (Thr180/Tyr182) and phosphorylated AKT (Thr308/Ser473) were determined using monoclonal antibodies (Cell Signaling Technology, Inc). Detection was performed with the enhanced chemiluminescence after incubation with a suitable secondary antibody conjugated to horseradish peroxidase (ECL; Cell Signaling Technology). Densitometric analysis was performed using Quantity One software (Biorad).

Data Analysis
Data were expressed as means ± standard error of the mean (SEM). Hemodynamics data were analyzed with One-way analysis of variance (ANOVA) to determine differences between multiple groups. If an analysis of variance showed a significant difference, post hoc analysis with Tukey test was used for further comparisons. P<0.05 was considered statistically significant.

Results
The role of exogenously administered and endogenously produced Ang II in I/R injury and PPC was examined using recombinant Ang II and ACE and chymase enzyme specific inhibitors, respectively. Exogenously administered Ang II did not affect cardiac hemodynamics (Fig  2), infarct size or cardiac enzymes (Fig 3 and Table 1) in hearts subjected to I/R injury or PPC suggesting that exogenous administration of Ang II does not worsen I/R injury nor attenuates PPC mediated protection of isolated rodent hearts. In the heart Ang II can be produced locally by ACE-dependent and independent pathways. We therefore tested the involvement of locally produced Ang II in I/R injury and in PPC mediated cardioprotection. The involvement of ACE dependent and chymase mediated intracellular production of Ang II were tested by infusion of captopril or chymostatin at reperfusion, respectively. Myocardial infusion of captopril or chymostatin significantly (P<0.05) protected the heart against I/R injury (Fig 4) and reduced infarct size and cardiac enzymes (Fig 3 and Table 1). The protective effects of PPC on heart hemodynamics, infarct size and cardiac enzymes (Fig 3 and Table 1) however were not augmented by the administration of captopril or chymostatin (Fig 4). All together this data suggest the possible involvement of ACE and chymase dependent production of Ang II during I/R induced cardiac injury while the lack of an additive effect of captopril and chymostatin to PPC mediated protection may however suggest the existence of a common downstream effector pathway. Locally produced Ang II however does not seem to play a significant role in PPC induced protection.
Detrimental effects of Ang II in the cardiovascular system were shown to be mediated by AT1. We have therefore tested the involvement of AT1 in the observed effects of locally produced Ang II using Ang II receptor blocker, irbesartan. We anticipated that blockage of AT1 would reduce I/R injury and may potentially enhance PPC mediated cardiac protection. To our surprise however, infusion of AT1 receptor antagonist did not protect the heart against I/R injury and interestingly revoked the protective effects of PPC on coronary vascular dynamics (Fig 2), infarct size and cardiac enzymes (Fig 3 and Table 1).
To investigate possible downstream signaling pathways involved in PPC mediated cardiac protection we tested the effect of Ang II, ACE inhibitor, chymase inhibitor or AT1 antagonist in presence or absence PPC on the activation state of signaling mediators of the reperfusion injury salvage kinase (RISK) pathway. Ang II treatment did not affect the basal ratio of phosphorylated to total extracellular regulated kinases 1/2 (ERK1/2) or protein kinase B/Akt during I/R injury neither it blocked enhanced phosphorylation of ERK1/2 and Akt in response to PPC (Fig 5). Captopril and chymostatin however significantly (P<0.05) increased ERK1/2 and Akt phosphorylation during I/R injury, yet did not augment PPC-induced phosphorylation of ERK1/2 and Akt possibly suggesting RISK downstream of a common signaling pathway activated by PPC and by the inhibition of ACE and chymase (Fig 5). Blockade of AT1 did not induce ERK1/2 and Akt phosphorylation during I/R injury, however it completely blocked their activation during PPC (Fig 5). This observation is consistent with the lack of an effect of AT1 blockade on cardiac hemodynamics, infarct size and cardiac enzymes levels during I/R injury and elimination of PPC mediated protection. Finally no changes were observed in the phosphorylation state of p38 MAPK during PPC in the presence or absence of Ang II, captopril, chymostatin and irbesartan (data not shown) suggesting that p38 MAPK is dispensable in PPC mediated protection and in PPC interaction with RAS.

Discussion
In this study we tested the interaction between ACE-Chymase-Ang II-AT1 and PPC mediated cardiac protection in isolated rodent hearts. The main findings of this study are: (1) systemically infused Ang II does not enhance I/R injury nor abrogate the protective effects of PPC. (2) inhibition of ACE or chymase protects against I/R injury yet does not block or augment PPC mediated protection. (3) blockade of AT1 does not protect against I/R injury yet evokes the protective effects of PPC on coronary hemodynamics, infarct size and cardiac enzymes. (4) the interaction between ACE-chymase-Ang II-AT1 and PPC involves signaling via ERK1/2 and Akt members of the RISK pathway (Fig 6). The RAS is a key regulatory system of cardiovascular physiology in which Ang II is a major effector peptide [13]. Ang II can be produced systemically via ACE dependent pathway or locally via ACE-dependent or independent enzymes [20,21]. The exact role of RAS in I/R induced injury and PPC mediated ex vivo cardioprotection is however not clearly understood. Local production of Ang II by ACE and chymase is stimulated by stress and ischemic insult [22] and has been implicated in post ischemic cardiac remodeling [30]. Increasing body of evidence indicates the presence of ACE and supports its role in the intracellular production of Ang II in cardiomyocytes [31,32], heart failure [33] and myocardial infarction [34]. Enhanced chymase mediated intracellular production of Ang II was reported in the vasculature, isolated cardiomyocytes [35], atherosclerotic lesions [36] and infarcted myocardium [22]. The role of ACE or chymase stimulated production of Ang II in postconditioning remains however to be characterized. In this study we demonstrate that blockade of ACE or chymase mediated endogenous production of Ang II, by captopril and chymostatin respectively, alleviates I/R injury. Captopril mediated protection could be due to reduced production of endogenous Ang II or reduction in kinins degradation [37,38]. This finding sheds light on previously undiscovered role of ACE-dependent and ACE-independent (chymase mediated) endogenous production of  Pacing Postconditioning and RAS Ang II in I/R injury and highlights the existence of potentially, overlooked, targets in the clinical application of postconditioning. Administration of chymase inhibitors to animal models of atherosclerosis, hypertension and heart failure demonstrated multiple beneficial effects including reduced atherosclerotic plaque development [39], improved LV contractility [40] and attenuated post-MI cardiac remodeling with overall improved survival [4]. Furthermore oral administration of chymase inhibitor reduced cardiac injury in a dog model of I/R injury [7]. It cannot be ruled out however that the protective effects of captopril and chymostatin could due to their indirect effects on other peptides besides Ang II [41,42]. This possibility however remains to be further investigated.
In the cardiovascular system many of the physiological and pathophysiological effects of Ang II are mediated by AT1 receptors [30]. We therefore tested the role of AT1 in PPC   Fig 6. Proposed mechanism of the interaction between ACE-Chymase-Ang II-AT1, I/R injury and PPC mediated cardiac protection. Ischemia/ reperfusion induced cardiac injury characterized by impaired cardiac hemodynamics, increased infarct size and enhanced release of cardiac enzymes, LDH and CK, involved ACE and chymase mediated production of Ang II and reduced activation of ERK1/2 and AKT components of the RISK pathway. PPC on the other hand improved cardiac hemodynamic and attenuated infarct size and release of cardiac enzymes. PPC protection involved downstream signaling via the RISK pathway. Exogenous administration of Ang II or blockage of endogenous Ang II had a neutral effect on PPC mediated cardiac protection yet induced the RISK pathway. AT1 antagonist, irbesartan on the other hand abrogated the protective effects of PPC and attenuated the RISK pathway.
doi:10.1371/journal.pone.0165777.g006 mediated cardiac protection. Based on our data from captopril or chymostatin treated hearts, we anticipated that inhibition of AT1 will improve the protective effects of PPC. To our surprise however, PPC mediated protection on cardiac hemodynamic and infarct size were lost upon the infusion of AT1 antagonist (Fig 6, Table 1). This may suggest the possible requirement of AT1 signaling in PPC mediated protection of rodent hearts ex vivo. Signaling via AT1 involved G-protein dependent and G-protein independent pathways [22]. While G-protein signaling has been linked to I/R induced damage, G-protein independent signaling was shown to induce cardioprotection [43]. Simultaneous blockade of G-protein coupled signaling and activation of G-protein independent signaling through AT1 receptor protected against I/R induced injury as indicated by reduced infarct size [43]. Another possible explanation to the loss of PPC mediated cardiac protection in the presence of AT1 antagonist could be due to mechanical stretch induced changes in AT1 signaling and/or localization. Mechanical stretch was reported to activate AT1 receptor independent of Ang II [44]. In addition AT1 was reported to undergo a conformational switch in response to mechanical stretch resulting in inverse agonist-induced inactivation [45]. Tension induced thinning of the membrane lipid bilayer has been suggested as a possible mechanism. However the precise mechanism by which mechanical force is transmitted to AT1 remains to be identified [45]. Similar inverse behavior was shown in α2A-adrenergic receptor whereby inverse agonists resulted in a conformational rearrangements in the receptor and subsequent suppression in activity [46]. Thus its plausible to speculate that pacing induced mechanical stretch may alter AT1 mediated signaling whereby the AT1 antagonist (irbesartan) acts as a receptor agonist. However, this possibility remains to be tested.
The RISK is a cascade of kinase that has classically been implicated in cardioprotection. The pathway involves parallel activation of PI3K/Akt and ERK1/2 upon engagement of g-protein coupled receptors or growth factor receptors resulting in inhibition of glycogen synthase kinase 3β and prevention of subsequent opening of mitochondrial permeability transition pores [47]. Activation of RISK family members ERK1/2 and Akt were shown to confer cardioprotection during reperfusion [48] and postconditioning maneuvers [49][50][51]. Here we show that PPC induced cardiac protection also involves enhanced activation of ERK1/2 and Akt (Fig 5). In contrast however Pipicz and his group have demonstrated that rapid ventricular pacing does not induce ERK1/2 activation [52]. The reason behind this discrepancy is however not clear. Differences in the pacing procedures used could be a possibility. In addition we demonstrate that the activation state of ERK1/2 and Akt is subjected to regulation by RAS during I/R injury and in PPC-induced protection. Consistent with its effects on cardiac hemodynamics, infarct size and cardiac enzymes levels, exogenously infused Ang II did not change the phosphorylation state of ERK1/2 and Akt during I/R injury and PPC. Inhibition of locally synthesized Ang II by the administration of captopril or chymostatin however significantly induced ERK1/2 and Akt phosphorylation during I/R injury yet did not enhance their activation in hearts subjected to PPC. Suggesting the potential involvement of locally produced Ang II in I/R induced injury and the possible convergence of protection induced by blockage of intracellular synthesis of Ang II and PPC into a common downstream signaling pathway that ultimately stimulates ERK1/2 and Akt. Finally blockade of AT1 did not alter the activation state of ERK1/2 or Akt in response to I/R injury. Inhibition of AT1 however significantly reduced the activation of ERK1/2 and Akt in response to PPC. Likewise this data is consistent with the lack of PPC induced cardiac protection in the presence of irbesartan.
Our study provided insight into ACE-Chymase-Ang II-AT1 role in I/R induced injury and PPC mediated cardiac protection. Findings from this study are likely to have a direct impact on our understanding of the physiological role of RAS in I/R injury and on RAS components that could potentially be attractive pharmacological targets. Our data suggests that AT1 antagonist (irbesartan) offsets the protective effects of PPC. This suggests that hypertensive patients on irbesartan or possibly other AT1 blockers need to be carefully considered in future PPC studies. Henceforth care must be taken when planning studies including this group of patients. Drug interference with postconditioning maneuvers was reported in patients with acute coronary syndrome and was suggested to interfere with postconditioning afforded protection [53], [54]. Our data further suggests that blockade of endogenously synthesized Ang II by captopril or chymostatin post-ischemically might represent a novel pharmacological alternative to PPC for the treatment of myocardial I/R injury as the attained cardioprotection was comparable to that of PPC. The latter finding may have important clinical implications as these drugs are already used in clinical practice and could potentially be safely administered after myocardial infarction. The use of ACE and chymase inhibitors in the treatment of myocardial infarction however needs to be further evaluated at pre-clinical and clinical levels.

Conclusions
In conclusion this study identifies a previously undiscovered role ACE and chymase induced synthesis of endogenous Ang II in mediating I/R cardiac injury and highlights the significance of ACE and chymase blockage as a potential therapeutic target in the treatment of myocardial infarction. It further characterizes a novel role of AT1 in PPC mediated cardiac protection of rodent hearts ex vivo. Finally it demonstrates that the RISK pathway is a downstream target of PPC and ACE-chymase-Ang II-AT1 signaling.


extfieldsofstudy: Biology Medicine

text:
Glucagon receptor gene deletion in insulin knockout mice modestly reduces blood glucose and ketones but does not promote survival

Objective It has been thought that the depletion of insulin is responsible for the catabolic consequences of diabetes; however, evidence suggests that glucagon also plays a role in diabetes pathogenesis. Glucagon suppression by glucagon receptor (Gcgr) gene deletion, glucagon immunoneutralization, or Gcgr antagonist can reverse or prevent type 1 diabetes in rodents suggesting that dysregulated glucagon is also required for development of diabetic symptoms. However, the models used in these studies were rendered diabetic by chemical- or immune-mediated β-cell destruction, in which insulin depletion is incomplete. Therefore, it is unclear whether glucagon suppression could overcome the consequence of the complete lack of insulin. Methods To directly test this we characterized mice that lack the Gcgr and both insulin genes (GcgrKO/InsKO). Results In both P1 pups and mice that were kept alive to young adulthood using insulin therapy, blood glucose and plasma ketones were modestly normalized; however, mice survived for only up to 6 days, similar to GcgrHet/InsKO controls. In addition, Gcgr gene deletion was unable to normalize plasma leptin levels, triglycerides, fatty acids, or hepatic cholesterol accumulation compared to GcgrHet/InsKO controls. Conclusion Therefore, the metabolic manifestations associated with a complete lack of insulin cannot be overcome by glucagon receptor gene inactivation.

INTRODUCTION
Hyperglucagonemia is present in many forms of diabetes [1,2] and contributes to elevated blood glucose by promoting glycogenolysis, gluconeogenesis, and ketogenesis while inhibiting glycogen synthesis. Remarkably, mice with whole body Gcgr gene deletion (GcgrKO) are protected from streptozotocin (STZ) induced diabetes [3,4]. While GcgrWT mice developed severe hyperglycemia, hyperketonemia, and cachexia and had to be euthanized 6 weeks post-STZ treatment, GcgrKO mice remained healthy [3,4]. Moreover, other studies have shown that glucagon suppression by Gcgr gene deletion, glucagon immunoneutralizing antibody, or Gcgr antagonism can reduce fasting and fed blood glucose and improve glucose tolerance in healthy and diabetic models [5e8]. These studies support the idea that elevated glucagon action is required for hyperglycemia in insulin-deficient type 1 diabetes. However, those studies used immune-or chemical-mediated destruction of b-cells that reduce but do not eliminate insulin. In those models, animals retain islet insulin immunoreactivity and circulating insulin, and survive for weeks without treatment [3e6,8]. As mice with knockout of both insulin genes (Ins1KO/Ins2KO, herein referred to as InsKO mice) survive for <2 days after birth [9] and approximately 1 day in adulthood following cessation of insulin therapy [10], it is evident that these other models are not insulin-free. Therefore, we aimed to determine whether loss of glucagon action improves glucose metabolism and promotes survival in the complete absence of insulin. To achieve this, we characterized mice with Gcgr, Ins1 and Ins2 gene deletions.

Animals
Mice with global Gcgr knockout, originally described by Gelling et al. [7], were supplied by Dr. Daniel Drucker. Male GcgrKO/Ins1KO/Ins2Het and female GcgrHet/Ins1KO/Ins2Het mice were bred to generate male and female experimental and control mice. Mice were genotyped using primers found in Supplemental Table 1. For pup characterization, InsKO pups were not treated with insulin. For young adult characterization, InsKO mice were treated twice daily with subcutaneous injections of Lantus insulin glargine (Sanofi-Aventis Inc., Laval, Canada). On P13-15, InsKO mice underwent a transplantation of w130 islets into the anterior chamber of the eye from w20 week old Ins1 À/À donors with 0e2 alleles of Gcgr and 1e2 alleles of Ins2. At 4 weeks of age mice underwent enucleation and were carefully monitored for rapid weight loss, hunching, piloerection, lethargy, and nonresponsiveness in order to euthanize any animals that reached humane endpoint. Mice were housed on a 14:10 h lightedark cycle with ad libitum access to food (5053, PicoLab Rodent Diet 20, LabDiet) and water. All experiments were approved by the University of British Columbia Animal Care Committee and performed in accordance with the Canadian Council on Animal Care guidelines.

Metabolic measurements
All parameters were measured in the ad libitum fed state. Blood glucose was measured via the tail vein using an OneTouch Ultra Glucometer (LifeScan) with a detection limit of 1.1e33.3 mM. In Figure 3G & H, one glucometer reading was above the limit of detection and assigned a value of 33.3 mM and statistical analysis was not performed. Plasma b-hydroxybutyrate (b-Hydroxybutyrate LiquiColor Test, Stanbio, Boerne, TX), glucagon (Glucagon ELISA, Mercodia, Salem, NC), leptin (Mouse Leptin ELISA, Crystal Chem, Downers Grove, IL), triglycerides and glycerol (Serum Triglyceride Determination Kit, SigmaeAldrich, Oakville, Canada), free fatty acids (HR Series NEFA HR [2] Kit, Wako Diagnostics, Richmond, VA), and cholesterol (Cholesterol E, Wako Diagnostics, Richmond, VA) were measured from trunk samples in pups or cardiac puncture samples in young adult mice which were collected as mice reached humane endpoint. Hepatic lipid extraction was performed as previously described [11].

Statistics
Data were analyzed using a 1-way or 2-way ANOVA with Bonferroni post-hoc testing for bar and line graphs respectively, or a Gehane BresloweWilcoxon test for survival curves. Statistical analysis was performed using GraphPad Prism 6.05 (La Jolla, CA). Significance was set at P < 0.05.

RESULTS
3.1. Effect of Gcgr gene deletion on survival in InsKO pups First, we aimed to determine whether Gcgr gene deletion would extend survival of InsKO pups. All male and female pups compared had 0 Ins1 alleles, 0e1 Gcgr alleles and 0e2 Ins2 alleles. GcgrHet/InsWT, GcgrKO/InsWT, GcgrHet/InsHet, and GcgrKO/InsHet pups were used as controls, while GcgrHet/InsKO and GcgrKO/InsKO pups tested the effect of Gcgr gene deletion on InsKO pups. All controls survived and gained weight throughout the first week of life, while both GcgrHet/ InsKO and GcgrKO/InsKO failed to gain mass and died at a similar rate, surviving no longer than 6 days ( Figure 1A & B). Therefore Gcgr gene deletion does not promote survival of InsKO pups.

Effect of Gcgr gene deletion on glucose and lipid metabolism in InsKO pups
We assessed if loss of glucagon action in InsKO pups improved metabolism by analyzing metabolic parameters associated with glucose metabolism in P1 pups. Results were variable, likely a reflection of litter size, when the animals last fed, and the time of day the samples were collected. Gcgr gene deletion with the InsWT or Het genotype did not affect any of the parameters measured. At P1 GcgrHet/InsKO pups weighed 30% less than controls, which was marginally increased in GcgrKO/InsKO pups but still 20% less than the control groups ( Figure 2A). As expected, GcgrHet/InsKO pups were hyperglycemic compared to controls. While still hyperglycemic, this was modestly decreased by 24% in GcgrKO/InsKO pups ( Figure 2B) relative to GcgrHet/InsKO pups. Similarly, plasma b-hydroxybutyrate levels were 9-fold higher in GcgrHet/InsKO pups compared to controls and reduced in GcgrKO/InsKO pups to 4-fold that of controls ( Figure 2C). It has been published that adult GcgrKO mice exhibit elevated glucagon levels [3,7]. At P1, glucagon levels were significantly higher than controls in GcgrKO/InsKO pups ( Figure 2D). Therefore Gcgr gene deletion modestly improved hyperglycemia and hyperketonemia in InsKO pups. Since type 1 diabetes is associated with aberrant lipid metabolism, we characterized lipid homeostasis in P1 pups. As expected, due to the positive effect of insulin on adipogenesis and leptin production [12], GcgrHet/InsKO pups had undetectable plasma leptin levels which were unchanged due to Gcgr gene deletion ( Figure 2E). Plasma triglycerides and free fatty acids were increased in GcgrHet/InsKO pups relative to Finally, hepatic cholesterol accumulation was unaffected by Gcgr gene deletion, and hepatic triglyceride levels in InsKO pups were comparable to that of controls ( Figure 2J & K). Therefore, Gcgr gene deletion does not normalize lipid metabolism in InsKO pups.

Effect of Gcgr gene deletion on survival of adult InsKO mice
We have shown that GcgrKO/InsKO pups do not have extended survival; however, because insulin is involved in growth and development [13], we sought to determine the effect of glucagon suppression in adult InsKO mice. As previously published, male and female InsKO mice were maintained to adulthood using insulin injections and islet transplantation into the eye [10]. GcgrKO/InsKO mice failed to grow normally following weaning despite post-transplant euglycemia ( Figure 3A & B). Despite receiving the same number of islets, GcgrKO/ InsKO mice had normal blood glucose levels compared to hyperglycemic GcgrHet/InsKO mice, suggesting that the loss of Gcgr aids in improving hyperglycemia when insulin is present. On day 0 of the study, the eye containing the exogenous islets was enucleated to render the InsKO mice insulin deficient; InsHet mice underwent enucleation to control for the effect of surgery. Following enucleation, all InsKO mice rapidly lost weight, were hunched, displayed piloerection, were lethargic or non-responsive, and reached humane endpoint at a similar rate, by day 6 of the study ( Figure 3C). As shown in Figure 3DeF, the kinetics of this weight loss was similar between GcgrHet/InsKO and GcgrKO/InsKO mice. Therefore in young adult mice, Gcgr gene deletion was unable to prevent body weight loss and death associated with a complete loss of insulin.
3.4. Effect of Gcgr gene deletion on glucose and lipid metabolism in adult InsKO mice We next sought to determine whether Gcgr gene deletion improved blood glucose levels in young adult InsKO mice. InsKO mice rapidly became hyperglycemic upon removal of the graft. At day 0.75, 1.25, and 1.75, blood glucose of GcgrKO/InsKO mice was modestly lower than that of GcgrHet/InsKO mice (Figure 3GeK). Interestingly, at humane endpoint, 3 of the GcgrKO/InsKO mice were hypoglycemic (<1.3 mM), which was not observed in the GcgrHet/InsKO mice ( Figure 3K and data not shown). In conclusion, Gcgr gene deletion modestly reduced blood glucose throughout the study and even resulted in hypoglycemia in some mice, but did not permit survival in young adult InsKO mice. Finally, we measured various metabolic parameters in young adult InsKO mice with and without Gcgr gene deletion. Similar to P1 pups, GcgrKO/InsKO mice had substantially elevated plasma glucagon levels compared to all other groups, while the GcgrKO/InsHet group trended towards increased levels of glucagon compared to GcgrHet groups ( Figure 4A). As expected b-hydroxybutyrate levels were substantially to that of controls, however there was large variability in ketones levels in the GcgrHet/InsKO mice ( Figure 4B). Plasma cholesterol levels were similar between controls and InsKO mice, and fatty acids levels were increased in GcgrKO/InsKO compared to controls ( Figure 4C & D).

DISCUSSION
Recent evidence suggests that glucagon itself causes the lethal catabolic consequences of insulin deficiency [14]. Remarkably, despite a 90% reduction in insulin, GcgrKO mice remained euglycemic and healthy compared to controls, which required euthanization 6 weeks  superscripts (a, b, c) are significantly different from each other within each graph. Statistical analysis was not performed on G and H as one blood glucose value fell above the limit of detection and was assigned a value of 33.3 mM, or in K because the n value decreased over time. Data are graphed as mean AE SEM (A, B, DeI), % survival (C), or individual mice (K), n ¼ 4e7, throughout study InsKO mice reached humane endpoint therefore n is reduced in E, F, H, and I. post-STZ [3,6]. However, we found that with 100% elimination of insulin, Gcgr gene deletion in mice does not extend survival. At both P1 and in early adulthood, GcgrKO/InsKO mice failed to gain mass or lost a substantial amount of weight and survived no longer than GcgrHet/ InsKO controls. In a previous study, we generated InsKO mice on a GcgrWT background and found that these mice survived for w1 day following islet graft removal, less than the 6 days observed in the current study. While it may appear that the reduction of one Gcgr allele may have had a slight protective effect on InsKO mice, we believe this is unlikely, because it has been shown that GcgrWT and GcgrHet mice display similar Gcgr function and metabolic phenotypes. Indeed, liver membranes prepared from GcgrWT and GcgrHet mice exhibit identical responses to glucagon stimulation as measured by cAMP production and these mice exhibit similar blood glucose elevations in response to i.p. injection of glucagon, suggesting that the loss of 1 allele does not significantly alter the function of glucagon receptor in the mouse [7]. In addition, in another strain of GcgrKO mice, it has been shown that gross appearance, body weight, visceral fat, liver and pancreas weight, and random glucose are not different between GcgrWT and GcgrHet mice [15]. Therefore, we believe that the difference in days of survival between GcgrWT and GcgrHet mice between studies may be due to differences between background strain, age, diet, and facility. Nonetheless, it is clear that deletion of both Gcgr alleles cannot promote long-term survival in mice completely lacking insulin as all reached humane endpoint within 1 week of birth. The loss of glucagon action in the complete absence of insulin does moderately improve aspects of metabolism. Glucagon plays a role not only in the fasting state but also in the fed state, which is evident as Gcgr gene deletion can normalize fed blood glucose levels and glucose tolerance [3,4,6]. In our study, GcgrKO/InsKO mice only experienced a partial improvement in the fed blood glucose levels in both P1 pups and young adults. Furthermore, although we saw modest blood glucose reduction throughout the study in the fed state, at euthanasia, some of the GcgrKO/InsKO were hypoglycemic, a state not observed in the GcgrHet/InsKO mice. The feeding state of the mice at the time of euthanization is unclear. Therefore, we speculate that if we had fasted the GcgrKO/InsKO mice, they may have experienced a larger decrease in blood glucose due to insufficient endogenous glucose production. Furthermore, it has been shown that Gcgr gene deletion normalizes ketones [3]. Since GcgrHet/InsKO mice had highly variable ketones, levels only trended towards improvement in GcgrKO/InsKO adult mice, and although we observed a significant improvement in ketones in P1 pups, levels in GcgrKO/InsKO mice still remained 4Â higher than controls. Interestingly, in both pups and adults, loss of the Gcgr gene did not improve aberrant lipid metabolism in the InsKO mice. Thus, while loss of glucagon action can improve some aspects of metabolism, metabolism is not normalized in the absence of both insulin and glucagon action. While several reports indicate that loss of glucagon action can protect rodents from diabetes [3e6,8], some studies suggest a less substantial role for glucagon in diabetes pathogenesis [16,17]. Meek et al. suppressed glucagon action in STZ-diabetic rats using a glucagon-like peptide 1 receptor agonist or a glucagon neutralizing antibody, both of which did not change blood glucose levels, but lowered ketones levels, suggesting that hyperglucagonemia is required for ketosis but not hyperglycemia [16]. Their report only partially agrees with our data as we observed minor improvements in both blood glucose and ketones. Furthermore, Steenberg et al. report that ablation of glucagon expressing cells using GCG-DTR mice does not improve glucose tolerance but modestly prevents further deterioration in STZ-diabetic mice [17]. In addition, a recent study by Damond et al. attempted to maximize insulin blockage in GcgrKO mice through combined STZ treatment and insulin receptor antagonism or diphtheria toxin induced b-cell loss [18]. Interestingly, although insulin-depleted GcgrKO mice were hyperglycemic compared to untreated GcgrKO mice, these mice had modestly improved blood glucose levels compared to GcgrHet mice treated with the same insulin-blocking regimen [18]. This effect is similar to the results of our paper, strengthening the conclusion that Gcgr gene deletion can only modestly reduce blood glucose levels in the absence of insulin but cannot prevent diabetes. Furthermore, in the report by Damond et al., although GcgrKO treated mice had decreased survival compared to untreated GcgrKO mice, they survived 3 times longer than GcgrHet mice with the same insulin-blocking regimen [18]. This is in contrast to our results in which both GcgrKO/InsKO and GcgrHet/InsKO mice survived for only 6 days. This difference may reflect the incomplete depletion of insulin by Damond et al., compared to the complete lack of insulin in our model. Collectively, we have shown that the absence of glucagon signaling can modestly improve diabetic symptoms in insulin knockout mice, but this improvement was insufficient in promoting survival. Therefore, we have provided unequivocal evidence that it is not possible to protect from the catabolic consequences due to the complete loss of insulin by eliminating glucagon action.