The co-crystal structure of the metallopeptidase astacin with its specific protein inhibitor fetuin-B reveals a novel mechanism of inhibition. XGP motif. The CY1 module assists in structural integrity and the CTR is not involved in inhibition, as verified by in vitro studies using a cohort of mutants and variants. Overall, the inhibition conforms to a novel ‘raised-elephant-trunk’ mechanism for MPs, which is reminiscent of single-domain cystatins that target cysteine peptidases. Over 200 sequences from vertebrates have been annotated as fetuin-B, underpinning its ubiquity and physiological relevance; accordingly, sequences with conserved CPDCP- and QWVXGP-derived motifs have been found from mammals to cartilaginous fishes. Thus, the raised-elephant-trunk mechanism is likely to be generally valid for the inhibition of astacins by orthologs of fetuin-B.Mammalian fetuin-A and fetuin-B are abundant serum proteins with pleiotropic functions. Fetuin-B is a highly selective and potent inhibitor of metallopeptidases (MPs) of the astacin family, which includes ovastacin in mammals. By inhibiting ovastacin, fetuin-B is essential for female fertility. The crystal structure of fetuin-B was determined unbound and in complex with archetypal astacin, and it was found that the inhibitor has tandem cystatin-type modules (CY1 and CY2). They are connected by an exposed linker with a rigid, disulfide-linked ‘CPDCP-trunk’, and are followed by a C-terminal region (CTR) with little regular secondary structure. The CPDCP-trunk and a hairpin of CY2 form a bipartite wedge, which slots into the active-site cleft of the MP. These elements occupy the nonprimed and primed sides of the cleft, respectively, but spare the specificity pocket so that the inhibitor is not cleaved. The aspartate in the trunk blocks the catalytic zinc of astacin, while the CY2 hairpin binds through a QWV This family is derived from the archetypal reversible inhibitor specific for cysteine peptidases, the monomeric 116-residue chicken egg-white cystatin  and CTR (residues F266–P388) domains of mouse fetuin-B and point mutants  of mouse fetuin-B, were expressed in ExpiCHO-S cells  according to the manufacturer’s specifications  and CTR (residues P254–I345) domains of mouse fetuin-A plus an intercalated mouse fetuin-B CY2 domain (residues V145–Q269) and was obtained from adenovirus-infected COS-7 cells  affinity chromatography as described previously  polyethylene glycol 2000, 0.1 M sodium acetate pH 4.6 as the reservoir solution. Crystals were cryoprotected by rapid passage through drops containing increasing concentrations of glycerol [up to 10%(v/v)]. It is noteworthy that properly diffracting crystals appeared after 4–5 days and rapidly lost diffracting power after a further 1–2 weeks. SDS–PAGE and N-terminal Edman degradation of washed and dissolved complex crystals revealed that fetuin-B was cleaved at positions S296–S297 within the CTR, which did not alter its inhibitory properties (data not shown).Crystallization assays of the astacin–fetuin-B complex were performed using the sitting-drop vapor-diffusion method. Reservoir solutions were prepared by a Tecan robot and 100 nl crystallization drops were dispensed onto 96 × 2-well MRC plates (Innovadyne) at the joint IBMB/IRB Automated Crystallography Platform at Barcelona Science Park using a Cartesian MicroSys 4000 XL robot (Genomic Solutions) or a Phoenix nanodrop robot (Art Robbins). Plates were kept in Bruker steady-temperature crystal farms at 4 or 20°C. Crystals were obtained by incubating the mouse inhibitor with a sixfold molar excess of the crayfish peptidase. The best crystals were obtained at 20°C in 0.1 µl:0.1 µl drops with protein solution at a concentration of 6 mg mlM sodium acetate, 25%(w/v) PEG 4000, 8%(v/v) isopropanol.Crystals of intact unbound mouse fetuin-B produced in HEK293S cells were grown using a Mosquito robot (TTP Labtech) by sitting-drop vapor diffusion at room temperature against a mother liquor consisting of 100 met al., 2014XDS  and diffracted to 3.1 Å resolution.Diffraction data for the astacin–fetuin-B complex crystals were collected at 100 K from liquid-nitrogen flash-cryocooled crystals (Oxford Cryosystems 700 Series Cryostream) using a PILATUS 6M detector (Dectris) on the XALOC beamline  on beamline ID29  plus S501–A502 from the C-terminal purification tag from molecule B and Q28–P388 plus S501  from molecule D. One N-acetyl-d-glucosamine (NAG) moiety was attached to N40 and N139 of molecule B, and one and two NAG moieties were linked to N40 and N139 of molecule D, respectively. Three glycerols plus 46 solvent molecules completed the model. Some regions of molecule D were only poorly defined in the final Fourier maps and were modeled based on molecule B to preserve the overall chain continuity, as this led to lower free R-factor values. The satisfactory quality of the final model was assessed with the wwPDB Validation Service . This model was expanded using AutoBuild within PHENIX. It was then manually rebuilt with Coot and refined against the first wedge of diffraction data with PHENIX. The final model included residues L31–P388 , one NAG attached to N40 and N139, one acetate and 85 solvent molecules. Structure validation was performed with MolProbity . Enzyme concentrations for astacin and meprin β were determined from the absorbance at 280 nm  and for ovastacin via the IC50 calculation of wild-type murine fetuin-B. Assays were performed in triplicate at 37°C in a 100 µl final volume, with 150 mM sodium chloride, 50 mM Tris–HCl pH 7.4, 0.01% Brij 35 as the buffer. Enzyme-activity measurements were started by the addition of 20–30 µM Ac-RE(Edans)DRNleVGDDPYK(Dabcyl)-NH2  for ovastacin and meprin β or 70–80 µM Dansyl-PKRAPWV-OH  for astacin dissolved in dimethyl sulfoxide . Initial velocities were recorded for at least 600 s . Thereafter, 1.5 µl proteinase K  or 1 µl astacin (at 200 µM) were added to reach complete substrate turnover, which was monitored and subsequently calculated using the formula v = [S] × m/ΔF, where [S] is the substrate concentration, m is the (F/t) slope of initial linear substrate turnover and ΔF is the maximal fluorescence intensity corresponding to complete turnover. Kinetic parameters of inhibition (Ki) were determined using Morrison’s equation . The area of the complex interface was taken as half of the sum of the buried surface areas of the molecules. Sequence-similarity searches were performed with the PSI-BLAST protocol at NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) or the BLAST protocol at UniProt (https://www.uniprot.org/blast) using default parameters. Sequence identities were calculated using SIM with default parameters (https://web.expasy.org/sim/). The final coordinates of the crayfish astacin–mouse fetuin-B complex and free fetuin-B are available from the PDB at http://www.rcsb.org .Sequences were aligned with 3.3.1.et al., 2012296–S297 within the CTR without affecting its inhibitory properties (data not shown). The resulting astacin–fetuin-B complex structure was solved by maximum-likelihood-scored molecular replacement, followed by density modification and noncrystallographic symmetry averaging, and was refined to 3.1 Å resolution . This introduces a crucial crystal-packing contact that would most likely be hindered by the presence of additional sugar residues attached to the core N-acetylglucosamine, such as those normally found in proteins expressed in insect cells.Several groups have unsuccessfully tried to crystallize fetuins since their discovery  in its astacin complex includes the domains and segments CY1 (R29–S146), linker  and CY2 , and S268–V272 plus P303–P388 from the CTR (S268–P388). The N-terminal 118-residue CY1 domain exhibits the cystatin fold and consists of a twisted and curled five-stranded antiparallel β-sheet (strands β1–β5) with simple up-and-down connectivity. An α-helix (α1) is inserted between β1 and β2 through short linkers and nestles perpendicularly to the strands in the concave face of the sheet [Figs. 1a) and 1b)]. Strand pairs β2/β3 and β4/β5 are connected by short loops and are arranged as hairpins I and II, respectively. In contrast, β3 and β4 are connected by a long 21-residue loop (Lβ3β4), which was found in several conformations in ovocystatin . A fifth cysteine (C39) in Lβ1α1 is bound to the CTR through a disulfide (see below), and two N-glycans are attached to N40 and N139, respectively. The C-terminal strand of CY1 (β5) contains a bulge and leads to the 12-residue LNK, which consists of a short α-helix (α2) and the segment C154PDCP158 arranged in a tight 1,4-turn of type I, hereafter referred to as the ‘CPDCP-trunk’ [Fig. 1c)]. Owing to the prolines and the cysteines, which are disulfide-linked (C154–C157), this structure forms a rigid 14-atom ring that protrudes from the molecular surface [Fig. 1b)]. In the absence of structural information, this disulfide had been previously termed type-C and included within the CY2 domain in fetuins and within the CY2 and CY3 domains in kininogen ]. Interestingly, the latter loop encompasses the segment L92ETDCHVLSRKA103, which has been identified as a characteristic signature of fetuins ].The LNK is followed by the 109-residue CY2 domain, which likewise adopts the cystatin fold and is topologically equivalent to the CY1 domain. It interacts through the convex face of its β-sheet with Lα1β2 and the legumain-binding loop from the CY1 domain . The respective cores comprising CY1+LNK+CY2 are undefined for both the tip of hairpin II and Lβ8β9 of CY2, and they superpose with an r.m.s.d. of 1.7 Å. CY1 and CY2 are just rotated by ∼10° with respect to each other when going from the unbound to the complexed structure. The only functionally relevant differences in the cores are found at the tips of hairpins I and II of CY2 [inset in Fig. 1e)], which however are very likely to result from crystal contacts in the unbound structure. Hairpin I is in a noncompetent conformation for binding, and becomes folded out towards the CPDCP-trunk upon complex formation. In addition, despite being disordered in both structures, hairpin II shows some extra ordered residues in the complex structure, possibly owing to fixing interactions with the target MP (see below). Although the CTR of unbound fetuin-B is intact and that of the complexed form is cleaved (see above), they show similar overall chain traces , as revealed by an r.m.s.d. of 1.3 Å upon superposition.The structure of unbound intact mouse fetuin-B is defined for residues L3.3.1–H202). It consists of an upper subdomain  and a lower subdomain  separated by an extended, deep, narrow active-site cleft  embedded in a long zinc-binding consensus sequence (H92EXXHXXGXXH102), which is a hallmark of the astacins , as well as another structural hallmark of astacins and other metzincins, the Met-turn . The C-terminus of the protein resides on the molecular surface and two disulfides (C42–C198 and C64–C84) contribute to the overall stability and rigidity of the molecule.Astacin is a 202-residue MP , 2b) and 2c)]. In contrast, CY1 makes only a minor contribution to binding and the CTR does not participate at all. Upon inhibitor binding, the LSD of astacin undergoes a slight clamshell motion of maximally ∼2.5 Å (at the Cα atom of Q118), which closes the active-site cleft as in the complex with an inhibitor mimicking a reaction intermediate ]. Fetuin-B segments involved include M111–Y113 from the legumain-binding loop of CY1, K147–S159 from the LNK and CY2 strand β6, N198–Y206 from CY2 hairpin I and, to a lesser extent, S245 plus Q253–V255 from CY2 hairpin II, the tip of which is disordered [see above and Figs. 2a) and 2c)].Mouse fetuin-B potently inhibits its physiological target ovastacin, human meprin β and archetypal crayfish astacin 156 in a bidentate manner, thus occupying the position of a scissile carbonyl during catalysis. The D156 carboxylate is also at binding distance from the zinc-binding atom Y149 Oη and, notably, the carboxylate of the general base/acid E93, so that either carboxylate could be protonated. P155 nestles into cleft subsite 2S .The main interaction is performed by the rigid CPDCP-trunk of the LNK, which blocks the nonprimed side of the cleft and binds the catalytic zinc through the side chain of Drger 1967 and Gomi al. 2012], C154 i199 side chain both positions the CPDCP-trunk through a hydrogen bond to D156 O and binds the main chain of the astacin bulge at S62 O. The downstream residue W200 makes a hydrophobic interaction through one face of its side chain with F154 and M124; through the other it is kept in place by the side chain of P204 upstream in the hairpin. The V201 side chain occupies 2′S and interacts hydrophobically with the sulfurs of the disulfide C64–C84, while S202 enters 3′S [Figs. 2c) and 2d)]. Owing to its missing side chain, the downstream residue G203 is essential to prevent a clash with W158 of astacin. Moreover, Y206 establishes a hydrogen bond to D121. Overall, these interactions are key for complex formation and comprise a motif for the tip of CY2 hairpin I (Q199WVSGP204), which is conserved as QWVXGP among mammalian orthologs ]. This impairs the functionality of the QWVXGP motif (H76YQEDMGS83 in CY1) and would cause collision with the primed side of the astacin cleft. Finally, CY1 hairpin II is seven residues shorter than in CY2, so that even the minor contribution to inhibition by this structural element is missing. To sum up, the CPDCP-trunk and CY2 hairpin I are the major determinants of inhibition, while CY1 and CTR merely play a scaffolding role.Overall, the CPDCP-trunk and CY2 hairpin I both bind in the direction of a substrate and cover the nonprimed and primed sides of the active-site cleft from 2bo9; Pallarès et al., 2005As to structurally similar inhibitors, human latexin likewise consists of tandem cystatin-like domains connected by a helical linker (PDB entry 3.4.a)], which add to previous studies  of selected constructs and mutants of mouse fetuin-B to provide quantitative data [Figs. 3b) and 3c)].To verify the abovementioned mechanism, we tested the inhibition of several constructs and mutants of human, mouse and bovine fetuin-B against crayfish astacin, human meprin β and mouse ovastacin [Fig. 3a)], in accordance with previous studies ]. In contrast, the Ki values of a chimera of mouse fetuin-B, in which CY1 and CTR were replaced by the homonymous domains of non-inhibitory mouse fetuin-A (mutant mFBABA), was decreased by more than three (astacin) and four (meprin β and ovastacin) orders of magnitude, respectively [Figs. 3b) and 3c)]. The mutants mFBBAA and mFBBAA could not be produced, which underpins the requirement for a complementary CY1–CTR interface for the proper folding of fetuin-B.The three full-length wild-type orthologs completely inhibited all enzymes [Fig. 3a)]. Taken together with the lack of inhibition of isolated domain CY2 [Fig. 3a)], this indicates that these modules are only functional within the context of a multi-domain fetuin. Domain CTR alone did not show inhibition either [Fig. 3a)], which is consistent with recombinant carp fetuin truncated after CY2 inhibiting meprin α/β and carp nephrosin with similar potency as the functional full-length protein purified from fish blood ], which indicates that the minimal functional structure does not require the overarching clamp between CY1 and CTR.A cyclic peptide including a sequence CPDC, which mimics the isolated CPDCP-trunk between CY1 and CY2, did not inhibit either enzyme and was indistinguishable from a variant with an arginine replacing the aspartate . Thus, similar to the mFBABA mutant, the double mutant is merely a micromolar inhibitor [Fig. 3c)] that is insufficient to block ovastacin under physiological conditions. Likewise, the C154S+C157S mutant led to a substantial decrease in Ki. Finally, grafting the sequence of CY1 hairpin I onto CY2  also led to substantially higher Ki values [Figs. 3b) and 3c)]. This indicates that the conserved QXVXGP sequence motif of H1 is required for efficient inhibition of astacins.Point mutations affecting the trunk within full-length mouse fetuin-B all had negative effects on inhibition. The POverall, the fact that the mutants and variants have comparable effects on astacin, meprin β and ovastacin supports the crayfish enzyme as a valid structural model for the mammalian orthologs. The results confirm that the major functional interactions between fetuin-B and astacin/ovastacin are made by the CPDCP-trunk and hairpin I of domain CY2 and that any modification leads to a decrease in inhibitory power. Moreover, an overall scaffold encompassing CY1+LNK+CY2 is indispensable to exert full inhibitory potency.3.5.1S and upstream nonprimed subsites of the cleft . Finally, hairpin II of cystatin provides a scaffold for hairpin I and performs ancillary interactions with the enzyme through the conserved PW motif. Overall, the inhibitory mechanism of MPs by fetuin-B, in which the extended N-terminal trunk is replaced by a compact CPDCP-trunk that resembles a ‘raising trunk’ [inset in Fig. 2d)], contains elements of similarity to the hydrophobic tripartite wedge of cystatins tackling cysteine peptidases. This suggests that gene duplication and molecular evolution of cysteine-peptidase targeting cystatins eventually led to inhibition of peptidases of a different chemical class. We thus hereby propose the term ‘raised-elephant-trunk mechanism’ to describe the mode of inhibition of astacins by fetuin-B [Fig. 2d)].The inhibitory mechanism of cysteine peptidases by monomeric cystatins has been dubbed the ‘elephant-trunk model’ ].The alignment of representative sequences of  mammalian fetuin-B orthologs revealed that they share the same overall length (374–388 residues) and show high sequence identity to the human form, which ranges from 98% (gorilla) to 47% (opossum)  contain potential orthologs with both the CPDCP-trunk and a putative hairpin I with a QWVe Table 3. They sh3.7.et al., 2019et al., 198922–S138; human fetuin A residue numbers are shown in italics; see UniProt P02765), LNK (A139–P150), CY2 (L151–Q251) and CTR (T252–V367) domains. It would also include a potential CPDCP-trunk (C146QDCP150), which would be solvent-exposed, as well as the upstream major physiological trypsin cleavage site of human fetuin-A , which could potentially interfere with active-site residues of the MP and thus impair inhibition. In contrast to the fetuin-B orthologs, the trunk motif shows variability across mammalian fetuin-A orthologs: CPRCP in mouse, CPQCP in cat, CPNCP in pig etc. In the bovine and rabbit orthologs the sequence even corresponds to that of fetuin-B. Collectively, these findings support the above results in vitro that the ‘warhead’ is a necessary but not sufficient element of inhibition. Indeed, the main reason for the lack of inhibition of human fetuin-A is that CY2 hairpin I is one residue shorter, shows a disparate sequence (V191PLPP195 instead of QWVXGP) and probably shows a distorted geometry owing to the two extra prolines [Fig. 2e)]. In addition, the CY2 hairpin II would be nine residues shorter (K237–G240 instead of S244–E256). Taken together, these differences in crucial and ancillary structural elements of the raised-elephant-trunk model would explain the lack of inhibitory competence of fetuin-A.The second position of the CPDCP-trunk is occupied by a glutamine in human fetuin-A (Q4.et al., 2010et al., 2012Discovered over seven decades ago in mammalian serum, the fetuins are a widespread family of vertebrate proteins. Fetuin-B is the only endogenous, specific, potent inhibitor of non-BMP-1-like astacin MPs through a conserved aspartate within a CPDCP-trunk that blocks the active-site cleft. This is reminiscent of the aspartate-switch mechanism that is responsible for latency in astacin zymogens, in which an aspartate likewise blocks the catalytic zinc (Guevara i.e. it can potentially adopt several conformations. By lining the exposed surface of CY1 and being covalently linked to it, the CTR provides fetuins with an adaptable surface to potentially bind many proteins and exert disparate signaling functions (Dunker et al., 2000CY1, LNK and CY2 are required for the inhibitory function of fetuin-B, but not the CTR, which is flexible and depleted of regular secondary structure, XGP motifs throughout jawed vertebrates down the tree of life as far as cartilaginous fish. In turn, astacins are present across animals, including lower vertebrates and invertebrates such as nematodes, mostly as several paralogs per organism (Gomis-Rüth, Trillo-Muyo et al., 2012Finally, we found sequences of proven and potential fetuin-B orthologs encompassing the CPDCP and QWVmouse fetuin-B, 6hpvPDB reference: complex with astacin, 6ht9PDB reference: 10.1107/S2052252519001568/jt5031sup1.pdfSupplementary figures.. DOI:


Insulin-induced gene (Insig) negatively regulates SREBP-mediated de novo fatty acid synthesis in the liver. However, the upstream regulation of Insig is incompletely understood. Here we report that AMPK interacts with and mediates phosphorylation of Insig. Thr222 phosphorylation following AMPK activation is required for protein stabilization of Insig-1, inhibition of cleavage and processing of SREBP-1, and lipogenic gene expression in response to metformin or A769662. AMPK-dependent phosphorylation ablates Insig’s interaction with E3 ubiquitin ligase gp78 and represses its ubiquitination and degradation, whereas AMPK deficiency shows opposite effects. Interestingly, activation of AMPK by metformin causes an augmentation of Insig stability and reduction of lipogenic gene expression, and leads to the attenuation of hepatic steatosis in HFHS diet-fed mice. Moreover, hepatic overexpression of Insig-1 rescues hepatic steatosis in liver-specific AMPKα2 knockout mice fed with HFHS diet. These findings uncover a novel effector of AMPK. Targeting Insig may have the therapeutic potential for treating fatty liver disease and related disorders. Insulin-related gene (Insig) negatively regulates hepatic fatty acid synthesis, a process involved in development of non-alcoholic fatty liver disease (NAFLD). Here, the authors show that AMPK activation by metformin promotes Insig phosphorylation, stabilizing it and inhibiting lipogenic gene expression. This is protective against steatosis in diabetic mice. SREBP is synthesized as precursor protein and retained in an inactive form in the endoplasmic reticulum (ER)2, where it is bound to two other proteins, SREBP cleavage-activating protein (SCAP) and insulin-induced gene (Insig)4. When the cellular cholesterol levels are low, the SCAP–SREBP complex dissociates from Insig, then transports from ER to Golgi, where SREBP is cleaved by two membrane-bound proteases in a process called regulated intramembrane proteolysis (RIP). The released NH2-terminal segment of SREBP translocates to the nucleus and stimulates lipogenic gene expression6.Nonalcoholic fatty liver disease (NAFLD) develops when aberrant triglyceride accumulation in the liver is not compensated by the increased rate of fatty acid expenditure. Excessive hepatic de novo lipogenesis plays an important role in the development of NAFLD. Sterol-regulatory element-binding protein (SREBP) is a key transcription factor that regulates fatty acid synthesis6. Insig-1 is highly expressed in the liver, whereas Insig-2a is a liver-specific transcript of Insig-26. Insig-1 and Insig-2 share similar function in that both isoforms cause ER retention of the SCAP/SREBP complex and exert a negative feedback control system on lipogenesis7. Transgenic overexpression of Insig-1 in the liver inhibits SREBP processing and lipogenesis8. In contrast, double knockout (DKO) of liver-specific Insig-1 and whole-body Insig-2 in mice  results in increased lipogenic program and dramatic accumulation of lipid in the liver9. In sterol-depleted cells, Insig-1 protein is ubiquitinated and rapidly degraded by E3 ubiquitin ligase gp78 with a half-life of less than 30 min10. Interestingly, proteasomal degradation of Insig-1 is at least 15 times more rapid than Insig-2 due to the serine residues flanking the sites of ubiquitination7. However, the upstream signaling that mediates the post-translational regulation of Insig is poorly understood.Insig is a potent inhibitor for the proteolytic process and maturation of SREBP via the retention of SCAP/SREBP complex in the ER11. Once activated, AMPK inhibits various anabolic pathways, stimulates catabolic pathways, suppresses ATP consumption, and increases ATP production to restore energy homeostasis13. We have previously identified that AMPK is a direct upstream kinase of SREBP. AMPK-dependent phosphorylation of SREBP-1c at ser372 site is sufficient and required for the inhibition of proteolytic cleavage and nuclear translocation of SREBP-1c14. However, SREBP-1c S372A mutation remains responsive to AMPK-mediated proteolytic cleavage and maturation of SREBP-1c, albeit the extent is less than wild-type (WT) SREBP-1c. These results suggest that additional AMPK substrates may directly or indirectly modulate SREBP-1c cleavage. Insig causes retention of the SCAP/SREBP complex in the ER, negatively regulates the cleavage of SREBP-1c, resulting in attenuation of lipogenic gene expression. However, whether AMPK regulates SREBP through Insig is not known.AMP-activated protein kinase (AMPK) monitors cellular energy status in response to nutritional variation in the environment15. Here, we provide insights into the mechanism by which AMPK inhibits cleavage and activation of SREBP-1c via phosphorylation. Gain-of-function and loss-of-function studies characterize Insig as a critical effector in mediating AMPK and its agonist metformin in regulating lipogenesis and maintaining hepatic lipid metabolism. These in vivo and in vitro studies characterize that (1) AMPK is an upstream kinase of Insig; (2) AMPK-dependent phosphorylation of Insig ablates its interaction with E3 ubiquitin ligase gp78; (3) Thr222 phosphorylation of Insig-1 is essential for AMPK to enhance Insig-1 activity and inhibit SREBP-1c proteolytic cleavage and target lipogenic gene expression; (4) Metformin attenuates hepatic steatosis in part through activation of Insig.We have recently identified transcriptional downregulation of Insig in the adaptive response to refeeding and under nutrient overload conditions through a novel metabolic cofactor CREBZF19. To investigate the efficacy and mechanisms of metformin on hepatic steatosis and lipid metabolism, 8-week-old male C57BL/6 mice were placed for 8 weeks HFHS diet, and followed by a dose of metformin (50 mg/kg/day), which has been shown to lower hepatic steatosis in high-fat diet-fed mice20. As shown in Fig. Several clinical studies indicate that the antidiabetic drug metformin might be of benefit for the treatment of NAFLD in humansTo investigate whether AMPK might be responsible for the protective effects of metformin, hepatic AMPK activity was assessed by determining the phosphorylation levels of AMPK and its downstream substrate acetyl-CoA carboxylase (ACC). As shown in Fig. To test whether AMPK regulates Insig in vitro, metformin was used to treat HEK293 cells transfected with plasmid encoding Insig. Real-time PCR analysis was performed to determine the effects of metformin on transcription levels of Insig-1. Surprisingly, mRNA levels of Insig-1 were not obviously changed by metformin  of protein degradation. To test this hypothesis, HEK293 cells were transfected with myc-tagged Insig-1 or FLAG-tagged Insig-2 together with or without HA-tagged ubiquitin (HA-Ub). As shown in Fig. 21 significantly reduced ubiquitination levels of both Insig isoforms. These data indicate that AMPK activation decreases ubiquitination levels of Insig.Given that regulated degradation of Insig is central for SREBP processing and cellular lipid homeostasis10, we focused on the study of AMPK’s effects on Insig-1 protein stability. HEK293 cells stably expressing Insig-1 were treated with cycloheximide, a eukaryote protein synthesis inhibitor, to measure the half-life of Insig-1. As shown in Fig. We next investigated whether AMPK regulates Insig degradation. Given that Insig-1 is degraded much more rapid than Insig-222, in which gp78 may also increase the degradation of both Insig-1 and Insig-2 through ubiquitination pathway. However, gp78 was characterized as an E3 ligase for Insig-1, instead of Insig-2, in Chinese hamster ovary (CHO) cells and human fibroblasts23. To investigate whether gp78 regulates expression levels of both Insig isoforms, HEK293 cells were transfected with Insig-1 and Insig-2 together without or with gp78. As shown in Fig. It has been reported that protein levels of both Insig-1 and Insig-2 are dramatically elevated in the liver of liver-specific gp78 knockout (gp78 LKO) mice fed with chow dietTo investigate whether AMPK-stimulated Insig expression is mediated via the regulation of expression levels of the ligase gp78, real-time PCR analysis was performed. As shown in Supplementary Fig. 24 had the opposite effect. Notably, phosphorylation levels of AMPK in response to A769662 and compound C were evidenced. These results demonstrate that Insig-1 is regulated by AMPK through phosphorylation.We hypothesized that AMPK might increase Insig activity though phosphorylation. To define Insig as a novel target of AMPK, Co-IP analysis was performed. As shown in Fig. We further investigated the AMPK phosphorylation sites in Insig-1. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis was performed and a few putative phosphorylation sites in human Insig-1 protein were revealed, including Ser43, Ser46, Ser74, Thr216, Thr222, and Ser276. Strikingly, the mutation of T222A-eliminated metformin-caused induction of Insig-1 Figs.  and S3B,25, it is technically difficult to determine whether the change of nuclear SREBP fragment is due to the regulation of proteolytic cleavage or transcription. We therefore sought to generate exogenous FLAG-tagged SREBP expression system, in which FLAG-tagged SREBP-1c is driven by a cytomegalovirus (CMV) promoter, which is not affected by autoregulation of SREBP. As shown in Fig. Next, we sought to study the mechanisms of AMPK on the proteolytic processing of SREBP-1c. Given that the positive feedback regulation of SREBP through the SRE element in its own promoter14. To investigate the relative contribution of Ser372 to AMPK-caused inhibition of SREBP-1c cleavage, HepG2 cells stably expressing nonphosphorylatable SREBP-1c S372A mutant were generated. As shown in Fig. It has been reported that AMPK phosphorylates SREBP-1c at Ser372 site to repress SREBP-1 proteolytic cleavage and attenuate hepatic lipogenesisTo further test the functional consequence of AMPK-SREBP-1c S372 mutant on hepatic lipid metabolism, oil red O staining was performed. As shown in Fig. 26. In contrast, AMPK inhibition by compound C caused a profound reduction of Insig-1 protein levels. To further investigate whether AMPK inhibition causes an increased degradation of Insig, cycloheximide assays were performed in HEK293 cell stably expressing Insig-1. AMPK inhibition by compound C significantly promoted the degradation of Insig-1, resulting in the half-life change of Insig-1 from 30 min in vehicle-treated cells to around 20 min in compound C-treated cells , hepatic AMPKα2 deficiency caused mild steatotic phenotypes in mice fed with HFHS diet as evidence by H&E staining, oil red O staining and triglyceride levels. Notably, the knockout efficiency of AMPKα2 specifically in the liver was verified by immunoblots. Strikingly, hepatic overexpression of Insig-1 rescued hepatic steatosis in AMPKα2 LKO mice. Next, we further determined whether reduction of SREBP-1c signaling and lipogenic program is responsible for improved hepatic steatosis in Ad-Insig-1-treated mice. The expression of Insig-1 in the liver was evidenced by RT-PCR  were performed in liver-specific AMPKα2 knockout (AMPKα2 LKO) mice. As shown in Fig. PCR Fig. . mRNA leThis report demonstrates that Insig may act as a novel substrate for AMPK in regulating hepatic lipogenesis and maintaining lipid metabolism through a post-translational modification. Metformin causes an AMPK-dependent activation of Insig through association and phosphorylation, which results in ablation of SREBP-1c proteolytic processing and activation, and attenuation of lipogenesis. Mechanistically, AMPK alpha subunit may directly phosphorylate Insig-1 at Thr222 site; and phosphorylation of Insig represses its ubiquitination and proteasomal degradation, and leads to increased protein stability and activity of Insig. AMPK-dependent phosphorylation of Insig may represent a molecular mechanism by which AMPK agonists, such as metformin ameliorate hepatic steatosis.One of the most important finding is that Insig-1 may be a novel substrate of AMPK. AMPK gain-of-function and loss-of-function approaches demonstrate that AMPK is sufficient and necessary for metformin, A769662 and AICAR on the stabilization and activation of Insig protein. Several evidences have demonstrated that Thr222 phosphorylation of Insig-1 is essential for AMPK to enhance Insig-1 activity and inhibit SREBP-1c proteolytic cleavage and target lipogenic gene expression. First, the phosphorylation of Insig-1 following AMPK activation was ablated by the nonphosphorylatable T222A mutant, indicating that Thr222 is specialized phosphorylated in an AMPK-dependent manner. Second, the T222A mutant, not the others, eliminated metformin-caused induction of Insig-1, suggesting that Thr222 site is essential for AMPK’s stimulating effects on Insig-1. Third, T222A mutant was resistant to A769662-caused protein stabilization, suggesting that AMPK-dependent phosphorylation at Thr222 site is essential for the stabilization of Insig-1. Fourth, T222A mutant diminished the inhibitory effects of A769662 on SREBP-1c cleavage. Moreover, the findings that the physical association between AMPKα subunit and Insig-1 is enhanced by AMPK agonist A769662, suggest that activation of AMPK may enable its substrates to be more accessible to the kinase domain of AMPKα, which led us to propose that Insig-1 may be a direct substrate of AMPK. Together, these data suggest that AMPK, via Thr222 phosphorylation, inhibits proteasome-mediated degradation of Insig-1, which results in a reduction of SREBP-1c cleavage processing and lipogenic gene expression. Interestingly, the mutation of S46A blocks metformin-induced Insig-1 levels in Fig. 14, the current study provides a novel mechanism of AMPK in regulating fatty acid synthesis via Insig. The human HepG2 hepatocytes stably expressing SREBP-1c WT or S372A mutant protein are of importance for the study of SREBP proteolytic processing and activation.Here we found that nonphosphorylatable SREBP-1c S372A mutant remains responsive to metformin-inhibited proteolytic cleavage in HepG2 hepatocytes, although the magnitude is lowered than WT SREBP-1c protein. Taken together with AMPK-dependent phosphorylation and inhibition of SREBP10. Moreover, activation of AMPK reverses the gp78-mediated degradation of Insig-1 likely via disrupting association between Insig and gp78. These data suggest that AMPK increases Insig stability by inhibiting gp78-mediated proteasome pathway. Proteasomal degradation of ubiquitinated protein is widely regulated by a phosphorylation-dependent mechanism. Phosphorylation of LIN28, a highly conserved RNA-binding protein, by MAPK/ERK increases its protein stability27. The proinflammatory kinase Inhibitor of nuclear factor κB kinase β (IKKβ) phosphorylates X-box-binding protein 1 (XBP1) inhibits its protein degradation mediated by ubiquitin proteasome pathway28. Interestingly, AMPK-dependent phosphorylation of thioredoxin-interacting protein (TXNIP) leads to its accelerated degradation and improved glucose uptake29. We report here that AMPK physically associates with Insig-1 to enhance Insig-1 protein stability via phosphorylation.The present study provides evidences that AMPK reduces proteasomal degradation of ubiquitinated Insig protein via gp78. First, AMPK activation by Metformin, A769662, or AICAR increases the protein levels of Insig and results in prolonged half-life. Second, AMPK represses ubiquitination and degradation of both Insig isoforms. Compared with Insig-2, Insig-1 is highly ubiquitinated, which is consistent with previous observation showing a much more rapid degradation of Insig-130, whereas during refeeding, Insig-2a is potently inhibited by a novel metabolic regulator CREBZF15. Together with the current findings showing post-translational regulation of Insig via AMPK, the dynamic regulation of Insig provides a finely tuned mechanism for the cellular regulation of hepatic lipid homeostasis.Recent studies demonstrated that the transcription of Insig-2a is regulated during the adaptive metabolic response to fasting or refeeding. During fasting, Insig-2a is induced by glucocorticoid-SETDB2 signaling31, and in humans19. We report here that metformin protects against hepatic steatosis in DIO mice. Beneficial effects of metformin on hepatic steatosis are at least partially mediated through AMPK-Insig pathway. First, metformin stimulates AMPK activity in the liver of HFHS diet-fed mice, which is positively correlated with increased expression levels of Insig-1 and Insig-2. Second, metformin increases the half-life of Insig-1 protein. Third, treatment with metformin reverses the gp78-mediated degradation of both Insig isoforms. Moreover, SREBP-1c S372A mutant remains responsive to metformin-caused reduction of SREBP-1c proteolytic activation and lipid accumulation in HepG2 cells, suggesting a potential role of the novel AMPK substrate Insig in mediating metformin’s effects in lipogenesis. These results identify Insig as a novel mediator in regulating metformin’s beneficial effects on hepatic lipogenesis and lipid metabolism. Further studies are needed to validate these mechanisms in humans.Metformin has long been recommended as first-line treatment in various guidelines for treating type 2 diabetes. Treatment with metformin has been shown to improve NAFLD phenotypes in genetically obese ob/ob mice8. In this study, moderate hepatic steatosis was observed in AMPKα2-deficient mice fed with HFHS diet, whereas previous reports showed that liver-specific AMPKα2 knockout mice fed with chow diet develop glucose intolerance in response to fasting without obvious hepatic steatosis32. The difference may be attributed to the different components of the diets, different pathophysiological conditions or different genetic backgrounds of the mice. Nevertheless, it would be interesting in future studies to address these questions. Together, these results support the notion that Insig plays a central role in regulating lipogenic gene expression and lipogenesis34, and that Insig may serve as a therapeutic target to ameliorate hepatic steatosis and related metabolic disorders.The in vivo and in vitro studies demonstrate that augmentation of Insig activity is efficient to inhibit hepatic lipogenesis and attenuate excessive lipid accumulation in hepatocytes and in livers. Insig activation by AMPK inhibits the cleavage of SREBP-1c and reduces lipogenic gene expression and lipid accumulation. Moreover, adenovirus-mediated overexpression of Insig-1 rescues hepatic steatosis in AMPKα2 LKO mice fed with HFHS diet, which is consistent with the reduced hepatic lipogenesis and triglyceride levels in the liver-specific Insig-1 transgenic miceIn summary, the current study identifies a biochemical mechanism of Insig regulation by AMPK Fig. . AMPK acflox/flox) were used as the control. Liver-specific AMPKα2 knockout (AMPKα2 LKO) mice were generated by crossing the floxed AMPKα2 mice with Alb-Cre recombinase transgenic mice, the WT littermates  were used as the control. Male C57BL/6 mice at 8 weeks of age were purchased from Shanghai Laboratory Animal Co. Ltd, China. Mice were placed on high-fat and high-sucrose (HFHS) diet 36 for 8 weeks, and then fed with HFHS diet supplemented with daily metformin (50 mg/kg/day) or vehicle (PBS) treatment via intraperitoneal injection for 8 weeks. All mice were housed under a 12:12-h light/dark cycle at controlled temperature. All animal experimental protocols were approved by Institutional Animal Care and Use Committee at Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.Liver-specific AMPKα1/α2 double knockout (AMPK−/−) mice were generated by crossing floxed AMPKα1/α2 mice, which was generated by crossing floxed AMPKα1 mice with floxed AMPKα2 mice , with albumin-cyclization recombination (Alb-Cre) recombinase transgenic mice ; WT littermates  were used for oil red O staining for the assessment of hepatic steatosis according to the manufacturer’s instructions .Livers were fixed in 10% phosphate-buffered formalin acetate at 4 °C overnight and embedded in paraffin wax. Paraffin sections (5 μm) were cut and mounted on glass slides for H&E staining35. Briefly, adenoviruses (5 × 109–1 × 1010 pfu per mouse) were delivered into mice by tail-vein injection. Two weeks post-injection, the mice were sacrificed in a postprandial state under isoflurane anesthesia, and tissues were rapidly taken and freshly frozen in liquid nitrogen and then stored at −80 °C or fixed for histologic analysis.Adenovirus-mediated gene transfer of Ad-Insig-1 or Ad-GFP in the liver of AMPKα2 LKO mice or WT littermates was performed38. Briefly, HEK293T cells were transfected with lentiviral transfer plasmid , along with packaging plasmids pMDLg/pRRE and pRSV-Rev (1:1 ratio), and envelope plasmid pMD2.G using polyethylenimine (PEI). The media containing lentiviral particles was stored at 4 °C. For lentivirus infection, HEK293 or HepG2 cells were cultured, treated with lentiviral particle solution, and then selected and passaged under puromycin-containing media for 4–7 days. The resulting cells stably expressing Insig or SREBP were ready for further assays.Lentiviral particles were generatedAMPK agonist metformin (cat. D150959), cycloheximide (cat. C1988), Oil Red O (cat. O0625), and mouse monoclonal FLAG M2 antibody (cat. F3165) were purchased from Sigma-Aldrich . A769662 (cat. sc-203790) was from Santa Cruz Biotechnology . AICAR (cat. A611700) was from Toronto Research Chemicals. Compound C (cat. 11967) was from Cayman Chemical . MG132 (cat. 1748) was from TOCRIS Bioscience . Glutathione-Sepharose beads (cat. 17-0756-01) were from GE Healthcare . Rabbit monoclonal phosphor-AMPKα (Thr172) antibody , rabbit monoclonal total AMPKα pan antibody , rabbit polyclonal Histone H3 antibody , rabbit monoclonal GAPDH antibody , mouse monoclonal myc-tag antibody  and rabbit monoclonal HA-Tag antibody  were obtained from Cell Signaling Technology . Rabbit polyclonal antibody against phospho-acetyl CoA carboxylase (Ser79)  was obtained from Merck Millipore. Total acetyl CoA carboxylase  was obtained from Upstate Biotechnology . Mouse monoclonal FAS antibody , mouse monoclonal phosphoserine/threonine (pSer/Thr) antibody  were from BD Biosciences . Antibodies against Insig-1 , Insig-2 , GST , SREBP-1 , β-actin ; horseradish peroxidase-conjugated anti-mouse  and anti-rabbit  secondary antibodies; and protein A/G PLUS-Agarose beads were obtained from Santa Cruz Biotechnology . The antibody against SREBP-2  was purchased from Abcam .39. To generate the plasmids encoding nonphosphorylatable S43A, S46A, S74A, S125A, S178A, S190A, S209A, T216A, T222A, S238A, S276A mutants of Insig-1, site-directed mutagenesis was carried out on myc-Insig-1. The following primers were used: for S43A mutant, AACGTTTCCGTGGCCGGGCCCTCCC (F) and GGGAGGGCCCGGCCACGGAAACGTT (R); for S46A mutant, TCCGGGCCCGCCCTGCTGGCG (F) and CGCCAGCAGGGCGGGCCCGGACA (R); for S74A mutant, CCCGAGCCCGGCGCCCCCTACCCCAA (F) and TTGGGGTAGGGGGCGCCGGGCTCGGG (R); for S125A mutant, CCATCTTTTCCGCCGCCTGGTGGGTCC (F) and GGACCCACCAGGCGGCGGAAAAGATGG (R); for S178A mutant, TAACCACGCCGCTGCTAAATTGG (F) and CAATTTAGCAGCGGCGTGGTTAATGC (R); for the S190A mutant, TGTCCAGCTGGCCTTGACTTTAGC (F) and TGCTAAAGTCAAGGCCAGCTGGAC (R); for S209A mutant, GATCGTTCCAGAGCTGGCCTTGG (F) and CCAAGGCCAGCTCTGGAACGATC (R); for T216A mutant, GCTAGAAAAGCTATGGCGATCCCCAGCCCAAGG (F) and CCTTGGGCTGGGGATCGCCATAGCTTTTCTAGC (R); for T222A mutant, GCGTGATCAGCGCAGCTAGAAAAGCTATGGTGATCC (F) and GGATCACCATAGCTTTTCTAGCTGCGCTGATCACGC (R); for T238A mutant, CTATCAGTATACAGCCCCAGATTTC (F) and AGAGGAAATCTGGGGCTGTATACTG (R); for T276A mutant, GTTCCTGAAAAGCCCCATGCTGATGGCGGCCG (F) and CGGCCGCCATCAGCATGGGGCTTTTCAGGAAC (R). Mutated bases were indicated by underlining. All constructs and mutations were verified by DNA sequencing. Transfection assays for plasmids were performed using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s protocol.The plasmid encoding FLAG-tagged human Insig-2 was constructed by amplifying PCR products from pCMV-Insig-2-myc  and inserting it into the EcoRI/XhoI site of FLAG-tagged pcDNA vector. The following primers were used: AATGCGAATTCATGGCAGAAGGAGAGACAGA (F) and AATGCCTCGAGTCATTCCTGATGAGATTTTT (R). The plasmid encoding HA-Ub has been described previouslyg for 5 min. Next, the cell pellets were washed with PBS, and centrifuged at 500×g for 5 min. To get the cytoplasmic extracts, the supernatant was removed and discarded, 200 μl ice-cold CER I was added to the cell pellet. The tube was vortexed vigorously on the highest setting for 15 s, then the tube was incubated on ice for 10 min. Eleven microliters of ice-cold CER II was added to the tube, followed by vortex and centrifuged at 16,000×g for 5 min. The supernatant containing the cytoplasmic fraction was transferred to a new tube. To get the nuclear extract, the pellet fraction was suspended in 100 μl ice-cold NER, the tube was vortexed vigorously on the highest setting for 15 s, and the sample was placed on ice and vortexing was continued for 15 s every 10 min for a total of 40 min. After last centrifuge at 16,000×g for 5 min, the supernatant containing the nuclear fraction was transfered to a new tube. The cytoplasmic and nuclear extracts were collected and used for Immunoblots.Nuclear and cytosolic extraction was performed using the NE-PER nuclear and cytoplasmic extraction kit (cat. 78833) obtained from Thermo Fisher Scientific. Briefly, HepG2 cells were treated by metformin for 24 h, harvested with trypsin–EDTA and then centrifuged at 500×Cell lysates prepared from myc-tagged Insig-1-transfected HEK293 cells were incubated with anti-myc-conjugated beads. The immunoprecipitated proteins were resolved by SDS–PAGE and stained with Coomassie Brilliant Blue dyes. Protein bands that corresponds to myc-Insig-1 from A769662-treated or untreated cells were excised and digested with trypsin buffer for 16 h at 37 °C. The resultant peptides were analyzed on an ultra-high performance liquid chromatography system (AMR) coupled to an Orbitrap Q Exactive mass spectrometer (Thermo Fisher Scientific). To generate an extracted ion chromatogram, the raw data were processed using Sequest and Proteome Discoverer (Thermo Scientific) software and directly analyzed using Proteome Discoverer .36. In brief, cells were homogenized and lysed at 4 °C in lysis buffer  Nonidet P-40, 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 5 μg/ml leupeptin, and 1 μg/ml pepstatin). Cell lysates were centrifuged at 16,000 ×g for 10 min at 4 °C, and the resulting supernatant was used for immunoblotting analysis. Protein concentrations in cell lysates were measured using Bio-Rad Protein Assay Dye Reagent. For immunoblotting, 20–50 μg of protein was separated by 8–10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and then electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes in a transfer buffer consisting of 25 mM Tris base, 190 mM glycine, and 20% methanol. The membranes were blocked with 5% non-fat milk in Tris-buffered saline with 0.1% Tween 20 (TBST) and incubated with specific antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Immunoblots were visualized by LumiGLO chemiluminescence detection kit . The intensity of bands was quantified using ImageJ . Relative phosphorylation levels were normalized to those of endogenous proteins and presented as the means ± SEM. For Co-IP, cell lysates containing overexpressed recombinant or endogenous proteins were incubated with specific antibodies and protein A/G Sepharose beads at 4 °C for overnight. The precipitates were washed three times with ice-cold lysis buffer. For GST pull-down analysis, cell lysates containing overexpressed glutathione S-transferase (GST) tagged fusion proteins or endogenous proteins were incubated with Glutathione Sepharose 4B beads  at 4 °C overnight and washed as Co-IP. The precipitates were then analyzed by immunoblots.Immunoblotting analysis was carried out35. Briefly, six-well plates were coated by rat-tail collagen type I  for overnight, and then washed by PBS for twice. Briefly, mice were anesthetized with sodium pentobarbital , and the portal vein was cannulated under aseptic conditions. The liver was perfused with ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) solution  and Hank’s balanced salt solution (HBSS) containing 0.075% collagenase type I (Sigma-Aldrich), 10 mg/ml DNase I (Sigma-Aldrich), 200 units/ml penicillin, and 200 μg/ml streptomycin, and then digested with 0.025% collagenase solution for the mouse liver. The isolated mouse hepatocytes were then cultured at 80–90% confluence in DMEM medium containing 10% FBS in rat-tail collagen type I-coated six-well plates (BD Biosciences) for overnight. Cells were infected with adenoviruses expressing Insig-1 or GFP, treated without or with metformin, A769662 and AICAR in a serum-free medium.Primary mouse hepatocytes were isolated40. Cells were cultured in DMEM containing 5.5 mM d-glucose,10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin, and incubated in a humidified atmosphere of 5% CO2 at 37 °C and passaged every 2 days by trypsinization. Cells were transfected with plasmids for 24 h, followed by treatment with 2 mM metformin for 24 h, 5 μM MG132 for 8 h, or 1 mM AICAR 24 h. For adenovirus infection, cells were treated with adenoviruses encoding constitutive active AMPK (CA-AMPK) or GFP for 48 h. SRD-13A cells (deficient in SCAP derived from CHO-7 cells)7 were generous gifts from Drs. Michael S. Brown and Joseph L. Goldstein at University of Texas Southwestern Medical Center, USA. The cells were grown in monolayer at 37 °C in 5% CO2, and were maintained in medium D. Medium D including 1:1 mixture of Ham’s F12 medium and Dulbecco’s modified Eagle’s medium containing 100 units/ml penicillin, and 100 μg/ml streptomycin, supplied 5% fetal bovine serum, 5 μg/ml cholesterol, 1 mM sodium mevalonate, and 20 μM sodium oleate41.Human HepG2 hepatocyte and HEK293 cells were purchased from Cell Bank, Type Culture Collection Committee, Chinese Academy of Sciences . The cells were cultured, and treated as indicated15.Liver tissues or cells were lysed in TRIzol Reagent (Life Technologies) to extract total RNAs according to the manufacturer’s protocol. Total RNAs were then reversely transcribed to cDNA using SuperScript II reverse transcriptase (Life Technologies) and Oligo d (T). The resulting cDNA was subjected to real-time PCR with gene-specific primers in the presence of SYBR Green PCR master mix (Applied Biosystems) using StepOnePlus Real-Time PCR System (Applied Biosystems)t-test and among more than two groups by analysis of one-way ANOVA. Differences were considered significant at a P-value < 0.05.Data are expressed as mean ± SEM. Statistical significance was evaluated using the unpaired two-tailed Student’s Supplementary InformationPeer Review FileReporting Summary


In comparison to other species, there are little data evaluating hoof conformation in dairy goats. As poor conformation is associated with an increased risk of hoof lesions and lameness, it is important to be able to accurately and reliably assess hoof conformation. This study developed a reliable hoof conformation assessment for use in dairy goats using hoof photographs. The assessment included both subjective scores and objective measures. High levels of accuracy were achieved when comparing two aspects of the subjective scores against two objective measures. This suggests the subjective scores may be a suitable alternative to the more time-consuming objective measures.The assessment of hoof conformation is important due to its recognized relationship with the biomechanical functionality of the hoof. Hoof conformation can be assessed using objective measures or subjective scores. However, to date, there are limited data using either method in dairy goats. Therefore, the aims were to (1) develop a reliable method of assessing hoof conformation in dairy goats, and (2) compare two aspects of a subjective assessment against corresponding objective measures as a means of validation. A total of 1035 goats contributed photographs across 16 commercial dairy goat farms. Photographs were taken of the left front and left hind hoof in the lateral and dorsal aspect at five assessments across the goats′ first two lactations. Hoof conformation was assessed using five subjective scores  and two objective measures (toe length ratio and claw splay distance). Following the training of two observers, high levels of inter and intra-reliability were achieved for both the subjective scores (>0.8 weighted kappa) and objective measures (>0.8 Lin′s concordance correlation coefficient). Two aspects of the subjectively assessed ordinal scores were compared with the objective measures with high levels of accuracy (>0.8). This suggests that the subjective scores may be a suitable alternative to more time-consuming objective measures when assessment is completed using photographs. Assessment of hoof conformation is important due to its recognized relationship with the biomechanical functionality of the hoof . Hoof coHoof conformation can be assessed using either subjective scores or objective measures. Aspects of the objective hoof conformation assessment described by Vermunt and Greenough  are ofteObjective measures are suggested to provide superior assessments compared to subjective scores as they are accurate and repeatable , allowinTo our knowledge, there is only one previous study that has objectively measured hoof conformation in dairy goats , which aSubjective assessments of hoof conformation involve visual assessments to allocate a categorical score for particular aspects of conformation ,6,11. ThIt is important that reliability testing is conducted for conformation scoring systems to ensure that accurate and reliable results are obtained. Without evaluating repeatability and reproducibility, any conclusions made from the results may be misleading . AdditioThis study was approved by AgResearch Ltd., Ruakura Animal Ethics Committee  as part of a large longitudinal study of dairy goat longevity. Sixteen commercial dairy goat farms in the Waikato region of New Zealand volunteered to participate . On 12 Farms were visited at five assessments throughout the goats′ first two lactations (2016–2017) . As partThe hoof conformation assessment was adapted from subjective scores and objective measures previously reported for several species . A digitThe assessment included five subjective scores: (1) toe length, (2) heel shape, (3) fetlock shape, (4) claw splay, and (5) claw shape . Each asTwo observers scored the photographs. Individual photographs were randomly allocated to each observer, ensuring that both observers scored photographs from each farm at each assessment. Observers completed scoring in a cyclical manner: A set of 20 photographs from one farm were completed and then the observer moved on to the next set to ensure photographs from several farms were scored on any given day. The subjective scoring and objective measures were performed in R 3.5.0 statistical software  . An R coUsing the developed code, a set of 20 photos were uploaded into R; the user firstly entered whether it was a lateral or dorsal aspect photograph they were viewing. A distance calibration was then completed using the scale bar marker on the whiteboard in the photographs. Four calibration points were selected on the scale bar. Two consecutive horizontal markers (x-distance) were firstly selected  and then two consecutive vertical markers (y-distance) were selected  a. The usFor the objectively measured toe length ratio, three points were selected on a lateral aspect hoof photograph; one point on the end of the toe (point 1), one point in line with the front edge of the coronet band (point 2), and one point at the back edge of the heel where the heel meets the ground (point 3) a. The diFor the claw splay distance, two points were selected on a dorsal aspect hoof photograph; one on the axial side of the distal tip of both claws, with the medial claw (inside claw) selected first (point 1) b. These Training involved scoring 400 photographs over 10 training sessions undertaken over a one-month period until an acceptable level of inter- and intra-observer reliability was achieved. A training session involved both observers independently scoring several photographs, and results were then compared and discussed before the next training session was conducted.Of the 13,921 hoof photographs scored in total, observer 1 scored 7901 and observer 2 scored 6020. The number of photographs scored by each observer contained an equal balance of both lateral and dorsal aspect photographs. Throughout the photograph scoring, on-going inter-observer reliability tests were completed after both observers had scored approximately 400 photographs. This resulted in 15 inter-observer reliability tests being completed. Intra-observer reliability was tested by observers re-scoring 10% of photographs from each farm at each assessment.wk) statistics were used to measure agreement. Acceptability was deemed as being above 0.8 , weighted kappa .For the objectively measured aspects of hoof conformation (toe length ratio and claw splay distance), the Bland–Altman method was used to graphically assess agreement . This inwk or CCC, further training was completed to ensure reliability was 0.8 or above before scoring of the photographs could continue.For each inter-observer reliability test, if reliability went below a threshold of 0.8 for either Data processing and descriptive statistical analysis was performed using R 3.5.0 statistical package . The objective measures of the toe length ratio and claw splay distance were checked for outliers. If data points were 3 or more times the interquartile range away from the first and third quartile, they were considered outliers. There were 40 photographs identified as outliers for the toe length ratio and 5 photographs identified for the claw splay distance. One observer rescored these photographs, and if the original measurement was accurate, the data point remained in the data set. After rescoring, 34 outliers were deemed as accurate for the toe length ratio and 4 for the claw splay distance and thus remained in the data set.To evaluate whether subjective scores were correctly assigned, thresholds were set for the toe length ratio as follows: If the ratio was <0.5 , the score = 0; if the ratio was >0.5 and <1 , the score = 1; and if the ratio >1 (length of the toe was greater than the full length of the rest of the hoof), the score = 2) . ThreshoContingency tables were produced to examine the assigned subjective scores for the toe length and claw splay to the actual scores  for the front and hind hooves across all assessments and farms. An overall accuracy was calculated for each of the ordinal categories  for the front and hind hooves. Accuracy was calculated at the level of each farm across the 5 assessments. Box plots were used to visually assess the consistency of scoring across the five assessments for the front and hind hooves.Accuracy was calculated as follows using the number of true positive (TP), true negative (TN), false negative (FN), and false positive (FP) :(3)Accurwk ranged from 0.32 to 0.86 . Over training sessions 5 to 7, wk ranged from 0.53 to 0.88 . From sessions 8 to 10, wk was consistently over 0.8. For the objective measures, over training sessions 1 to 4, CCC ranged from 0.52 to 0.79  for the toe length ratio and 0.24 to 0.95  for the claw splay distance. Over training sessions 5 to 7, the CCC ranged from 0.79 to 0.91  for the toe length ratio and 0.82 to 0.89  for the claw splay distance. Over training sessions 8 to 10, the CCC ranged from 0.79 to 0.92  for the toe length ratio and 0.93 to 0.95  for the claw splay distance.Over the 10 training sessions, inter-observer reliability increased. For the subjective scores over training sessions 1 to 4, The Bland–Altman plots for the measures of the toe length ratio and claw splay distance showed a random scatter of points, with the majority of points falling within the limits of agreement .wk) for the subjective scores and 0.76 to 0.99  for the objective measures throughout the study (wk threshold (0.63), and at test 8, the claw shape went below the 0.8 wk threshold (0.71)  (he study . At testd (0.71) .High levels of reliability were achieved for the fetlock shape subjective score; however, it should be noted that very few dipped fetlocks were recorded during the scoring of the lateral hoof photographs. A total of 186 were recorded out of 7058 lateral photographs .wk)) and the objectively measured aspects  (CCC)) of hoof conformation.Intra-observer reliability was consistently over 0.8 for the subjectively scored aspects  (High levels of accuracy were achieved for the subjective assessments of the toe length and claw splay (>0.8) for each of the ordinal score categories when compared with the objective measures. The accuracy was the highest when assigning a score of 0 and was lower for scores 1 and 2 for both toe length  and clawThe aim of this study was to develop a reliable method to assess hoof conformation in dairy goats. The results suggest that the assessment method developed is a suitable and reliable way to assess hoof conformation in dairy goats using photographs. After extensive training, both the subjective scores and objective measures were assessed reliably by the two observers. Two aspects of the subjective scores were compared with the corresponding objective measures and were found to be accurate. This suggests that the subjective scores, particularly the 0 and 2 scores, alone may be adequate to assess hoof conformation in dairy goats.Toe length, as a proxy for hoof overgrowth, is the aspect of hoof conformation that has previously been focused on in dairy goats ,25. ThisA potential limitation of subjective methods of hoof conformation assessment is the poor reliability between observers . PreviouTwo aspects of hoof conformation, toe length and claw splay, could be both subjectively scored and objectively measured, allowing comparisons between the two methodologies. When comparing the subjective scores and objective measures of toe length and claw splay, the observers in the present study were more accurate at assigning a score of 0 compared to 1 or 2, resulting in some overlap when looking at hooves with borderline scores. This highlights why a dichotomous score of “good” vs “bad” is commonly used in hoof conformation assessments. However, acceptable levels of accuracy (>0.8) were still obtained for scores 1 and 2 and this may be due to the intensive training that was completed prior to assessment of the photographs. We caution other authors that if an accuracy level of over 0.8 is required, collapsing scores to a binary assessment may be required. It should be noted that heel height has also been previously objectively measured in hoof conformation assessments in sheep and dairy cows, however, a lower observer reliability than other measurements of hoof conformation have been reported ,44; therThe present study highlights the need for considerable training to ensure inter- and intra-observer reliability when scoring hoof conformation from photographs. Intensive training was required to attain initial reliability levels and then ongoing reliability checks were conducted to ensure any deviation between the observers scoring was quickly detected. In contrast, Murray et al. (1994)  used thrwk and CCC. However, variance within an observer still needs to be reported. It is difficult to make definitive conclusions from studies where no evidence is provided to determine whether the method is repeatable or reproducible.Repeatability (intra-observer variation) and reproducibility (inter-observer variation) are important when trying to validate a method for assessing hoof conformation. However, for many hoof conformation assessments, repeatability and reproducibility have not been established. For example, Gomez et al. (2015)  evaluateHoof conformation has previously been objectively assessed using photographs with scale markers included for other species ,46, and The objective measure for the claw splay distance was consistently reliable throughout the scoring of the hoof photographs. There were two occasions when the reliability for the objective measure of the toe length ratio went below 0.8. This may have been due to difficulties in placing a point on the hoof in line with the front edge of the coronet band, especially if the hooves were particularly hairy or dirty. Due to time restrictions around milking and attempting to minimize the amount of time the goats were out of their pens, it was not feasible for the hooves to be washed. However, if possible, we recommend cleaning of the hooves prior to photographs being taken to improve reliability. As the reliability for the subjective score for toe length was consistently high throughout the assessments, it suggests that the subjective score is more appropriate to use rather than the time-consuming objective measure; however, this needs to be validated on farm.We successfully developed a reliable method for assessing hoof conformation in dairy goats using photographs. Two aspects of hoof conformation that were subjectively assessed were validated by the comparison of the subjective scores with objective measures. The use of photographs with scale markers allowed for objective measures to be completed; however, this was time consuming and required technical equipment. As two of the subjective scores were shown to correspond to objective measures, they are suitable methods for conformation assessment. High levels of accuracy and reliability (>0.8) were achieved on the photographs in this study; if higher levels were required, than collapsing the scores into a binary method should be considered. Nevertheless, further work is required to test the reliability and practicality of subjective hoof conformation assessment on live animals and to determine if it is applicable in an on-farm setting.


Failure of neural tube closure results in severe birth defects and can be induced by high glucose levels resulting from maternal diabetes. MARCKS is required for neural tube closure, but the regulation and of its biological activity and function have remained elusive. Here, we show that high maternal glucose induced MARCKS acetylation at lysine 165 by the acetyltransferase Tip60, which is a prerequisite for its phosphorylation, whereas Sirtuin 2 (SIRT2) deacetylated MARCKS. Phosphorylated MARCKS dissociates from organelles, leading to mitochondrial abnormalities and endoplasmic reticulum stress. Phosphorylation dead MARCKS (PD-MARCKS) reversed maternal diabetes-induced cellular organelle stress, apoptosis and delayed neurogenesis in the neuroepithelium and ameliorated neural tube defects. Restoring SIRT2 expression in the developing neuroepithelium exerted identical effects as those of PD-MARCKS. Our studies reveal a new regulatory mechanism for MARCKS acetylation and phosphorylation that disrupts neurulation under diabetic conditions by diminishing the cellular organelle protective effect of MARCKS. Neural tube defects can arise from high glucose levels caused by maternal diabetes, and MARCKS is required for neural tube closure. Here, Yang et al. show that acetylation and phosphorylation of MARCKS in hyperglycemic conditions causes mitochondrial and ER stress, leading to neural tube defects. The high glucose (HG) level in maternal diabetes induces NTD formation in both humans and animal models7. Mitochondrial dysfunction and endoplasmic reticulum (ER) stress in the developing neuroepithelium have been demonstrated to be critically involved in NTD formation in diabetic pregnancies;12 however, the mechanism underlying cellular organelle stress in diabetic embryopathy is unclear. During neurulation, neuroepithelial cells, which are essentially neural stem cells, undergo rigorous proliferation and migration to acquire the competence for neural plate elevation, neural fold convergence extension, and closure. Therefore, neuroepithelial cells may possess mechanisms for the protection of their organelles to ensure cellular homeostasis. Maternal diabetes may disrupt these mechanisms, leading to cellular organelle stress and NTD formation.Neurulation is a process occurring during early embryonic development in which the developing neuroepithelium is folded into the neural tube, which is the primitive form of the central nervous system (CNS). Failed neural tube closure leads to neural tube defects (NTDs), which are severe structural birth defects affecting offspring mortality and morbidityMarcks gene results in NTDs, mainly exencephaly13. MARCKS protein modification is critical for its biological function. Protein kinase C (PKC) phosphorylates MARCKS, which converts MARCKS from a membrane-bound protein to a cytoplasmic protein14. PKCs mediate the cellular stress response because deleting one of the PKC isoforms, i.e., the Prkcd gene, abolishes maternal diabetes-induced cellular organelle stress in embryos during the neurulation stage15. This evidence suggests that PKC triggers cellular stress by suppressing the protective effects of MARCKS on cellular organelle. However, the predicted cellular organelle protective effect of MARCKS has never been previously demonstrated.Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) is required for neurulation, and deleting the 17. The acetylation of a transcription factor can positively or negatively regulate the phosphorylation of the same protein, leading to increased or decreased activity of that transcription factor. Although MARCKS is not a transcription factor, it may be regulated by acetylation. In a large proteomic study, the lysine residue lysine 172 of human MARCKS was identified as an acetylation site18. Lysine 172 of MARCKS is adjacent to four serine residues that are often modulated by phosphorylation19. If MARCKS is regulated by acetylation, it could be interesting to determine whether MARCKS acetylation impacts its phosphorylation.Lysine acetylation is another protein modification that influences the biological action of proteins. The crosstalk between acetylation and phosphorylation in amino acid residues of the same protein has been revealed in transcription factors20. Various enzymes involved in histone acetylation, i.e., histone acetyltransferases (HAT), and histone deacetylation (HDAC), i.e., histone deacetylases, have been discovered. Currently, it is recognized that these histone enzymes also acetylate or deacetylate nonhistone proteins21. HATs and HDACs are substrate-specific22. If MARCKS is acetylated, a specific HAT should pair with an HDAC to regulate MARCKS acetylation. Among the HATs acetylating nonhistone proteins, Tat-interactive protein 60 (Tip60) is activated by cellular stress23 and induces DNA damage responses24, which are manifested in diabetes-induced NTDs, suggesting that Tip60 may mediate the cellular stress response in diabetic embryopathy by acetylating protein substrates. In contrast, the seven sirtuin HDAC family members (SIRT1-7) suppress cellular stress by deacetylating nonhistone proteins25. Therefore, we hypothesize that maternal diabetes induces cellular organelle stress by decreasing SIRT expression, thereby increasing the acetylation of their protein substrates.Since its discovery, histone acetylation has become a well-known euchromatin marker of the activation of gene transcriptionHere, we report that MARCKS is regulated by acetylation through Tip60 and SIRT2. We also show that maternal diabetes-induced MARCKS acetylation is required for its phosphorylation, which disables the protective effects of MARCKS on mitochondria and the ER, leading to cellular organelle stress and NTD formation. Preventing the phosphorylation of MARCKS under hyperglycemic conditions either with PD-MARCKS or SIRT2 overexpression restores the protective effects of MARCKS on the neuroepithelium. Thus, we provide mechanistic insight into the essentiality of MARCKS in neurulation.26. Because acetylation in a lysine residue of MARCKS has been detected in human tissues in a global acetylome analysis18, we sought to determine whether HG in vitro or maternal diabetes in vivo induces MARCKS acetylation. In the mouse neural stem cell line C17.2, HG levels mimic the key adverse effects of maternal diabetes, including cellular stress, apoptosis and histone acetylation, in neuroepithelial cells28. The HG levels triggered MARCKS acetylation in a dose-dependent and time-dependent manner in the C17.2 cells  is the putative acetylation site in mouse MARCKS  to prevent acetylation, serving as a dominant negative MARCKS (DN-MARCKS), or into Glutamine (Q) to mimic acetylation, thus serving as a constitutively active form of MARCKS (CA-MARCKS) 11 however, the underlying mechanism is unclear. Because MARCKS phosphorylation plays a key role in MARCKS cycling from phospholipid membranes, such as the plasma membrane, to the cytoplasm30, we hypothesize that MARCKS protects neuroepithelial cells from mitochondrial abnormalities and that phosphorylated MARCKS induced by hyperglycemia dissociates from the mitochondrial membrane and does not have a protective effect against the hyperglycemic insult exerted by mitochondria-bound MARCKS on cells. To test this hypothesis, mitochondria were purified from wild-type (WT) and MARCKS-PD (phosphorylation dead) transgenic embryos.Maternal diabetes induces mitochondrial abnormalities in neuroepithelial cells in the developing embryo;Under nondiabetic conditions, both WT MARCKS and MARCKS-PD were enriched in the mitochondrion Fig. . Materna11. Under the diabetic conditions, the overexpression of MARCKS-PD significantly reduced the number of defective mitochondria  pathways IRE1α and PERK were evaluated. The phosphorylation of IRE1α and JNK1/2 was induced by maternal diabetes. MARCKS-PD diminished the phosphorylation of these molecules Fig. . XBP1, w33. Thus, we examined the effect of MARCKS-PD on NTD formation. The NTD rate in the MARCKS-PD overexpressing embryos was significantly lower than that in the wild-type embryos from the diabetic dams and was comparable to that in the embryos from the nondiabetic dams  mouse line with MARCKS-PD overexpression in the developing neuroepithelium Fig. . As expeams Fig. . Thus, Mtes Fig.  and redutes Fig. .Fig. 5MA34. However, our previous study demonstrated that maternal diabetes delays neurogenesis in the developing neuroepithelium as evidenced by the absence of the expression of TuJ1 (class III β tubulin), which is a marker of terminal neuronal differentiation, from E8.75 to E9.0 and the enhanced expression of Sox1, which is a marker of neural stem cells11. Consistent with our previous findings, TuJ1-expressing neurons were absent, whereas Sox1 expression was enhanced in the E8.75 neuroepithelia from wild-type embryos exposed to maternal diabetes  assay was performed to evaluate the interaction between MARCKS and the three most common protein acetyltransferases, i.e., Tip60, Gcn5, and p300, using embryonic tissue extracts. Maternal diabetes enhanced the interaction between MARCKS and Tip60, whereas MARCKS-Gcn5 and MARCKS-p300 complexes were not observed  deacetylase family includes seven members (SIRT1-7) and regulates an array of cellular functions by primarily suppressing cellular stressCKS Fig. . The ectCKS Fig. . The SIRCKS Fig. . The recCKS Fig. . Deletinion Fig. . These d+/−) female mice were fed a pathogenic maternal diet37 and mated with SIRT2+/− male mice. The wild-type (WT), SIRT2+/−, and SIRT2−/− embryos all developed normally without NTDs 19 and the nonmyristoylated MARCKS mutant45. These findings suggest that membrane-associated MARCKS is required for, while cytoplasmic phosphorylated MARCKS may be detrimental to, CNS development.MARCKS was originally discovered as a major substrate of PKC in the CNS49. Both PKCα and PKCδ can induce MARCKS phosphorylation50. Increased MARCKS phosphorylation is implicated in the etiology of Alzheimer’s disease (AD)51, which is another neurological disorder. Amyloid beta, which is the toxic species responsible for AD pathology, causes MARCKS phosphorylation through PKC51. Our original hypothesis was that MARCKS phosphorylation is the pathological cause of the failure of neural tube closure in embryos derived from diabetic mothers. We observed that NTDs induced by maternal diabetes are inhibited by MARCKS-PD, supporting the causal role of MARCKS phosphorylation in failed neural tube closure in diabetic pregnancies.Under diabetic conditions, hyperglycemia activates PKCα and PKCδ, leading to NTD formation in the developing embryo52. MARCKS binds the membranes of mitochondria and the ER, and the phosphorylation of MARCKS caused by hyperglycemia causes it to dissociate from these intracellular membranes in embryos during the neurulation stage. Indeed, one proteomic study revealed enrichment in MARCKS in the mitochondrial fraction of neurons53. Once MARCKS binds the plasma membrane, it increases the abundance of the phospholipid phosphatidyl-inositol  bisphosphate in the plasma membrane54. The five phenylalanines in the MARCKS effector domain, in which the four PKC-phosphorylatable serines also reside, insert into the plasma membrane by further stabilizing the membrane54.MARCKS is associated with the cell plasma membrane and the intracellular membranes of mucin granules55. Phosphorylated MARCKS mimics the effect of MARCKS deficiency57. MARCKS phosphorylation leads to the loss of its function in protecting vascular endothelial cells from oxidative stress56. Thus, phosphorylated MARCKS is an inactive form of MARCKS. MARCKS phosphorylation under diabetic conditions abrogates its protection of cellular organelles.MARCKS likely maintains the integrity of and protects cellular organelle membranes from stress. During the neurulation stage, cellular organelles in embryos have been shown to be vulnerable to stress insults57. Nonphosphorylatable MARCKS abolishes, whereas pseudophosphorylated MARCKS mimics, PKC-induced neuron dysfunction58. This evidence suggests that MARCKS protects cells from stress, and its deficiency or phosphorylation causes cellular stress. Maternal diabetes-phosphorylated MARCKS dissociates from the mitochondrial membrane and the ER, leading to mitochondrial abnormalities and ER stress, which are two causal events in diabetic embryopathy27.MARCKS deficiency and phosphorylation mediate oxidative stress-induced vascular endothelial cell abnormalities59. Both the inadequate function of mitochondrial folate metabolism and the lack of key mitochondrial redox genes result in NTDs61. The overexpression of the mitochondrial antioxidant superoxide dismutase 2 ameliorates maternal diabetes-induced NTDs62. Here, we demonstrate that mitochondrial abnormalities due to MARCKS phosphorylation causes NTDs in diabetic pregnancies. MARCKS-PD blocks MARCKS phosphorylation-induced mitochondrial abnormalities by binding the mitochondrion and ER, leading to NTD reduction under diabetic conditions.Mitochondria are essential for embryogenesis64, suggesting that the IRE1α-ER stress pathway is highly relevant to diabetes-induced NTDs. ER stress is present in the neuroepithelia of embryos exposed to maternal diabetes, and the ER stress inhibitor 4-phenylbutyric acid reduces HG-induced NTDs in cultured embryos11. We showed that MARCKS-PD inhibits maternal diabetes-induced ER stress, supporting the protective effect of unphosphorylatable MARCKS on the ER.Normal ER function is required for neural tube closure. ER stress activates UPR signaling. The active form of XBP1, which is a major downstream effector of IRE1α, induces NTDs in XenopusMARCKS acetylation is required for its phosphorylation. The inhibition of MARCKS acetylation by SIRT2 blocks maternal diabetes-induced ER stress and NTD formation. Thus, maternal diabetes-induced MARCKS acetylation and subsequent phosphorylation disrupt the association between MARCKS and the ER and mitochondrion, leading to cellular organelle stress.39. The four serine residues in the effector domain of MARCKS, which have negative charges, bind the negatively charged phospholipids of the plasma membrane65. The N-terminal myristoyl group of MARCKS acts second by binding to the plasma membrane and facilitating a relatively stable interaction65.The phosphorylation and myristylation of MARCKS are well-recognized post-translational modifications that are primarily involved in the binding of MARCKS to or release from plasma membranesMARCKS on lysine residue 165 (K165) is important for its phosphorylation by PKC in neural cells. The phosphorylation sites in MARCKS PSD (phosphorylation site domain) are Serine 152 (S152), S156, S160 and S163. The acetylation residue K165 is adjacent to these four serine residues. K165 acetylation introduces another negative charge that may neutralize the positive charge in the nearby amino acid residues, leading to a MARCKS protein with a more open structure, particularly in the PSD. This structural change may render PKC more accessible to the four serine sites and potentiate these sites to becoming phosphorylated. However, directly investigating the physical function of acetylation on phosphorylation is very challenging. Future studies may explore these structural changes in the MARCKS protein upon acetylation.66. Phosphorylation plays a key role in cellular signaling, whereas acetylation functions in regulating protein expression and improving protein stability66. In some cases, the acetylation of proteins, especially transcription factors, enhances their transcriptional activity by increasing their DNA binding affinity, protein stability and phosphorylation sensitivity67. Similar to proteins, which become more sensitive to phosphorylation after they are first acetylated, acetylation is a prerequisite for MARCKS phosphorylation. Indeed, the MARCKS acetylation constitutive active isoform induces its phosphorylation under NG conditions. Moreover, the MARCKS acetylation dominant negative isoform blocks MARCKS phosphorylation increased by HG. Since the subcellular localization of MARCKS is controlled by its phosphorylation, MARCKS acetylation and resultant phosphorylation leads to a dissociation from cellular organelle membranes.We revealed that acetylation, which is another form of post-translational modification, regulates MARCKS phosphorylation under HG conditions. Protein function and subcellular localization are regulated by phosphorylation and acetylation57 however, the enzymes responsible for the acetylation/deacetylation of MARCKS are unknown. This study identified Tip60 as a MARCKS acetyltransferase and SIRT2 as a MARCKS deacetylase. Maternal diabetes in vivo and HG in vitro do not increase Tip60 expression but enhance its activity. In contrast, both SIRT2 expression and activity are downregulated by maternal diabetes. Further investigation indicated that restoring SIRT2 expression attenuated maternal diabetes-induced NTDs, suggesting that SIRT2-induced MARCKS deacetylation exerts a protective effect on neuroepithelial cells through the inhibition of MARCKS phosphorylation.Following acetylation, PKC and Protein Phosphatase 2A (PP2A) are responsible for the phosphorylation/dephosphorylation of MARCKS;39, identifying avenues, such as acetylation and phosphorylation, to modulate MARCKS activity and restore its functionality is fundamentally important.In summary, MARCKS acetylation, which is reversibly regulated by Tip60 and SIRT2, is a prerequisite for MARCKS phosphorylation, which is equivalent to loss of function mutations leading to NTDs, and mediates the teratogenicity of maternal diabetes in NTD induction by causing cellular organelle stress. Because MARCKS is broadly involved in an array of neurological disorders19 mice (C57BL/6J) were originally obtained from Dr. Perry J. Blackshear at the Signal Transduction Laboratory, National Institute of Environmental Health Sciences. The nestin promoter driven SIRT2 transgenic (SIRT2-Tg) mice were generated on a C57BL/6J background at the Genome Modification Facility, Harvard University. SIRT2 knockout mice were obtained from the Jackson Laboratory .The procedures used for the animal experiments were approved by the University of Maryland Baltimore Institutional Animal Care and Use Committee. The MARCKS-PD-transgenic (M-Tg)55 and other researchers69 have used a rodent model of Streptozotocin (STZ)-induced diabetes in research investigating diabetic embryopathy. Eight- to ten-week old female mice were intravenously injected with 65 mg/kg STZ daily into the tail vein over two days to induce diabetes. Diabetes was defined as 12-hour fasting blood glucose concentrations greater than or equal to 14 mM, which usually occurred 3–5 days after the STZ injections. We did not detect any differences in embryonic development between the STZ/insulin-treated and non-STZ-treated mice6, suggesting that STZ had no residual toxic effect in our animal model. The male M-Tg mice and SIRT2-Tg mice were bred with female diabetic mice. As previously described55, the embryos were harvested at E8.75 (2:00 PM at E8.5) for the biochemical and molecular analyses. At E10.5, the embryos were examined under a Leica MZ16F stereomicroscope  to identify the NTDs.We+/−) female mice were fed a Purina5015  or control  diet for 1 week prior to mating with the SIRT2 heterozygous (SIRT2+/−) male mice. To identify the NTDs, the embryos were examined at E10.5 under a Leica MZ16F stereomicroscope .Eight- to ten-week-old SIRT2 heterozygous (SIRT2+/−) female and male mice were mated overnight. The day on which a vaginal plug was observed was designated E0.5. At E8.5 (8 AM), mouse embryos with an intact visceral yolk sac were dissected from the uteri into PBS . Then, three embryos were cultured in 4 ml of culture medium containing 2.66 ml of rat serum, 1.34 ml of Tyrode's salt solution , 100 units/mL of penicillin and 100 µg/mL of streptomycin with or without 1 μM ROPA   at 38 °C in a roller bottle system. The embryos were cultured for 36 h under the following conditions: 5% O2−5% CO2–90% N2.The SIRT2 heterozygous  was used to detect apoptosis70. The C17.2 cells were cultured under NG or HG conditions and then transfected with scramble control siRNA or Tip60-siRNA  using Lipofectamine RNAiMAX  according to the protocol from the manufacturer. The C17.2 cells were transfected with pMARCKS-PD, pMARCKS-CA, pMARCKS-DN, pTip60, pSIRT2 and pPKCα-CA using Lipofectamine 2000  according to the protocol from the manufacturer.C17.2 mouse neural stem cells, which were originally obtained from the ECACC , are newborn mouse cerebellar progenitor cells transformed with retroviral v-mycIn total, 300 mg protein from C17.2 cells or five to six embryos pooled per litter, a protease inhibitor cocktail , lysis buffer , Protein A/G Magnetic bead slurry , and 1 mg rabbit anti-MARCKS antibody  were used for IP. The mitochondria were isolated from the C17.2 cells or embryos using a Pierce mitochondria isolation kit . For immunoblotting, equal amounts of protein 30 or 50 mg) from C17.2 cells or one whole embryo per dam were resolved by SDS-PAGE and transferred onto Immobilon-P membranes . Precision Plus Protein Standards  were loaded onto one lane of the gel. The membranes were incubated in 5% nonfat milk for 45 min, followed by incubation with the primary antibodies in 5% nonfat milk for 18 h at 4 °C. The detailed antibody information is provided in Supplementary Table 0 mg fromEmbryos at E8.5 were fixed in 4% paraformaldehyde (PFA) overnight, followed by embedding in optimum cutting temperature medium (OCT) compound . Then, 10-μm cryosections were pretreated with citrate buffer and blocked in 5% bovine serum albumin in PBST (0.1% Triton X-100 in PBS) for 1 h. The following antibodies were used as primary antibodies: MARCKS (1:200), TOM20 (1:200), Connexin (1:200), Sox1 (1:200), Tuj1 (1:200) and GFP (1:200). Normal rabbit or mouse IgG at the same dilutions as those of the antibodies were used as controls. After washing with PBS, the sections were incubated with the secondary antibodies. Then, the sections were counterstained with DAPI and mounted with aqueous mounting medium . The images were captured under a confocal microscope .E10.5 embryos were collected for a morphological examination. First, the embryos were fixed in methacarn , embedded in paraffin, and cut into 5-μm sections. After deparaffinization and rehydration, all specimens underwent hematoxylin and eosin (H&E) staining using a standard procedure. All embryo sections were photographed under a Nikon Ni-U microscope , and the NTDs were examined.The structures of the mitochondria were examined by transmission electron microscopy (EM) at our University EM core facility. Thick sections (1 μM) were cut and visualized at 100× magnifications to identify the neuroepithelium of the E8.5 embryos. Thin sections (80 nm) of the identified neuroepithelium were cut and viewed under an electron microscope  at a high-power resolution (6 and 15 K) for the identification of the cellular organelle structures.The mRNA of XBP1 was extracted from E8.5 embryos by TRIzol  and reverse transcribed to cDNA using a QuantiTect Reverse Transcription Kit . The PCR primers for XBP1 were as follows: forward, 59-GAACCAGGAGTTAAGAACACG-39, and reverse, 59-AGGCAACAGTGTCAGAGTCC-39. If no XBP1 mRNA splicing occurred, a 205-bp band was produced. If XBP1 splicing occurred, a 205-bp band and a 179-bp main band were produced. All primer sequences were listed in Supplementary Table t-test, and those in the comparisons of more than two groups were determined by one-way ANOVA using SigmaStat 3.5 software. In the ANOVA analyses, Tukey tests were used to estimate the significance of the results. Significant differences between the groups in the NTD incidences were analyzed by the Chi-square test. For all immunoblotting experiments, one embryo from one dam in each group was used for one run. Each experiment was repeated three times with three embryos from three different dams each group. For immunoprecipitation experiment, five or six embryos from one dam in one experimental group were combined and used for one run, each experiment was repeated three times with three different litters from different dams per group. All quantification data were indicated as means ± standard derivation.The statistical differences in the two group comparisons were determined by Student’s Supplementary InformationPeer Review File


Avian infectious bronchitis virus (IBV) frequently infects broilers and is responsible for severe economic losses to the poultry industry worldwide. It has also been associated with kidney damage in the broiler flocks. The aim of the present study is to determine the presence of IBV and its possible involvement in kidney damage of broiler chicks.14 clinically diseased broiler flocks from Western and Central Algeria were sampled and analyzed by hemagglutination inhibition (HI) test and reverse transcriptase-polymerase chain reaction (RT-PCR) followed by phylogenic analysis.The QX (100%) and 4/91 (60%) IBV serotypes were the most prevalent in the kidney damaged broilers regardless of vaccination status. The molecular detection of avian IBV by RT-PCR identified six samples as positive, of which only two isolates were typable by sequencing. We identified a novel IBDZ13a genotype which showed 93% sequence homology to the partial-S1 gene sequence of the IB 4/91 commercial vaccine strain. Sequencing analysis characterized this virus as a novel and divergent IB 4/91 field virus with eight amino acid substitutions that might have resulted in altered immunogenicity.The isolation of a new IBV strain (IBDZ13a) from vaccinated broiler flocks may explain the failure of the vaccination programs against IBV field strains. Combination of the HI test and RT-PCR indicated that the nephropathogenic IB outbreaks in broilers are related to this novel strain. Nidovirales and genus Coronavirus), frequently infects broilers and egg-laying hens and leads to severe economic losses to the poultry industry : 5’-TGA AAA CTG AAC AAA AGA CA-3’ and CK2 [reverse]: 5’-CTC GAA TTC CNG TRT TRT AYT GRC A-3’) were designed for the IBV S1 gene, which contains the hypervariable region 1 and most of the hypervariable region 2 . The S1 ®  Gel and PCR Clean-up kit according to the manufacturer’s instructions and sent for sequencing to x-OVO Limited . The nucleotide and amino acid sequences of the S1 gene of the IBV isolates were assembled, aligned, and compared with reference IBV strains using BASIC BLAST program in DNAStar. Phylogenetic analysis of the S1 amino acid sequences was performed with the neighbor-joining method using the DNAStar software [The amplified cDNA was purified using the NucleoSpinsoftware . The parThe specific IBV strains that were used in this study were M–41 (a US strain), 4/91 (European vaccine strain), D274 (a Dutch field isolate), and QX (Chinese strain). HI test was performed using the FLOKSCREEN IB-HI TEST kit according to the manufacturer’s instructions. Briefly, the sera were serially diluted and mixed with an equivalent volume of the antigen. After 30 min of incubation at 20°C, the red blood cells of SPF chickens (anticoagulant solution) were added and incubated further for 40 min at 20°C. The plates were then tilted to observe inhibition of hemagglutination.Statistical Package for the Social Sciences version 20  was used for statistical analysis. The geometric mean titer (GMT) was calculated by counting the number of wells from all serum samples within one flock showing HI activity, and the average number was then cross-checked against GMT values given in Brug’s table. The results were reported as the GMT for a given flock against a particular IBV antigen. Descriptive statistics including percentages, means, and frequency distribution were calculated for each of the variables.Information regarding the sampling date, biosecurity status, region of sampling, vaccination programs, necropsic and clinical signs, the age of the broilers, and mortality rates are summarized in 2) regardless of the vaccination status. Furthermore, 60% and 50% of the broiler flocks were seropositive for antibodies against the 4/91 and M–41 strains, respectively, although with low mean titer levels. The seroprevalence of IBV D–274 was the lowest with only 7% of the broiler flocks testing positive.The seroprevalence of various IBV strains in broiler flocks suffering from kidney damage in Algeria is summarized in 1=22.15 and Ct2=31.4). One of the two sequences was identified as the IBDZ13a genotype. Furthermore, IBV was only detected in the kidney tissues and not in the trachea. All samples showed 93% homology to the partial-S1 sequence acquired from the IB 4/91 reference strain  are the most commonly used tests for detecting IBV antibodies ,23. Studst time in Algeria by our group. After reporting the nephropathogenic form of IB [The most frequently detected IBV serotypes in the broiler flocks with nephritis were IB-QX (100%) and IB 4/91 (60%), which have been detected for the 1rm of IB , the aborm of IB ,28. The rm of IB  reportedrm of IB .et al. [et al. [et al. [The S1 protein of IBV determines the genotype, serotype, and phenotype of IBVs . The S1 et al.  and Kann [et al. , who cla [et al. . However [et al.  who also [et al. . Howeveret al. [When live-attenuated and inactivated vaccines of IBV were first introduced, they were effective in controlling IB among poultry flocks . Howeveret al.  demonstret al. ,36.st time in Algerian broiler flocks. Its isolation from the vaccinated flocks indicates poor protection afforded by the vaccination programs to the broilers against infection with new IB variants. Further studies are needed to explain the failure of the current vaccines against the new IB4/91 strain and to investigate the cross-protection and pathogenicity of the new genotype.We identified IBDZ13a, a new nephropathogenic variant of IB 4/91, for the 1AL and MO planned and designed the whole study. AL and HM collected samples, performed technique, and analyzed the data. KO and MO supervised the project and helped during manuscript writing, crosschecking, and revision. All authors read and approved the final manuscript.