Milk production may involve a transient development of insulin resistance in non-mammary tissues to support redistribution of maternal macronutrients to match the requirements of the lactating mammary gland. In the present study, adipose and liver metabolic responses were measured in the fasting state and during a 2-step (10 and 20 mU/m²/min) hyperinsulinemic-euglycemic clamp with stable isotopes, in 6-week postpartum women who were lactating (n=12) or formula-feeding (n=6) their infants and who were closely matched for baseline characteristics (e.g., parity, body composition, intrahepatic lipid). When controlling for the low insulin concentrations of both groups, the lactating women exhibited a fasting rate of endogenous glucose production (EGP) that was 2.6-fold greater, and a lipolysis rate that was 2.3-fold greater than the formula-feeding group. During the clamp, the groups exhibited similar suppression rates of EGP and lipolysis. In the lactating women only, higher prolactin concentrations were associated with greater suppression rates of lipolysis, lower intrahepatic lipid and plasma triacylglycerol concentrations. These data suggest that whole-body alterations in glucose transport may be organ specific and facilitate nutrient partitioning during lactation. Recapitulating a shift toward noninsulin-mediated glucose uptake could be an early postpartum strategy to enhance lactation success in women at risk for delayed onset of milk production.

The increasing prevalence of type 2 diabetes poses a major challenge to societies worldwide. Blood-based factors like serum proteins are in contact with every organ in the body to mediate global homeostasis and may thus directly regulate complex processes such as aging and the development of common chronic diseases. We applied a data-driven proteomics approach, measuring serum levels of 4,137 proteins in 5,438 elderly Icelanders and identified 536 proteins associated with prevalent and/or incident type 2 diabetes. We validated a subset of the observed associations in an independent case-control study of type 2 diabetes. These protein associations provide novel biological insights into the molecular mechanisms that are dysregulated prior to and following the onset of type 2 diabetes and can be detected in serum. A bi-directional two-sample Mendelian randomization analysis indicated that serum changes of at least 23 proteins are downstream of the disease or its genetic liability, while 15 proteins were supported as having a causal role in type 2 diabetes.

Reduction of β-cell mass and function is central to the pathogenesis of type 2 diabetes. The terms glucotoxicity, lipotoxicity, and glucolipotoxicity are used to describe potentially responsible processes. The premise is that chronically elevated glucose levels are toxic to β-cells, that elevated lipid levels in the form of circulating free fatty acids (FFA) also have toxic effects, and that the combination of the two, glucolipotoxicity, is particularly harmful. Much work has shown that high concentrations of FFA can be very damaging to β-cells when used for in vitro experiments, and when infused in large amounts in humans and rodents they produce suppression of insulin secretion. The purpose of this Perspective is to raise doubts about whether the FFA levels found in real-life situations are ever high enough to cause problems. Evidence supporting the importance of glucotoxicity is strong because there is such a tight correlation between defective insulin secretion and rising glucose levels. However, there is virtually no convincing evidence that the alterations in FFA levels occurring during progression to diabetes are pathogenic. Thus, the terms lipotoxicity and glucolipotoxicity should be used with great caution, if at all, because evidence supporting their importance has not yet emerged.

Prevention of aberrant cutaneous wound repair and appropriate regeneration of an intact and functional integument require the coordinated timing of fibroblast and keratinocyte migration. Here, we identified a mechanism whereby opposing cell-specific motogenic functions of a multifunctional intracellular and extracellular protein, the receptor for hyaluronan-mediated motility (RHAMM), coordinates fibroblast and keratinocyte migration speed and ensures appropriate timing of excisional wound closure. We found that, unlike in WT mice, in Rhamm-null mice, keratinocyte migration initiates prematurely in the excisional wounds, resulting in wounds that have re-surfaced before the formation of normal granulation tissue, leading to a defective epidermal architecture. We also noted aberrant keratinocyte and fibroblast migration in the Rhamm-null mice, indicating that RHAMM suppresses keratinocyte motility but increases fibroblast motility. This cell context–dependent effect resulted from cell-specific regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation and expression of a RHAMM target gene encoding matrix metalloprotease 9 (MMP-9). In fibroblasts, RHAMM promoted ERK1/2 activation and MMP-9 expression, whereas in keratinocytes, RHAMM suppressed these activities. In keratinocytes, loss of RHAMM function or expression promoted epidermal growth factor receptor–regulated MMP-9 expression via ERK1/2, which resulted in cleavage of the ectodomain of the RHAMM partner protein CD44 and thereby increased keratinocyte motility. These results identify RHAMM as a key factor that integrates the timing of wound repair by controlling cell migration.

Over the last several years it has become clear that higher order assemblies on membranes, exemplified by signalosomes, are a paradigm for the regulation of many membrane signaling processes. We have recently combined two-color direct stochastic optical reconstruction microscopy (dSTORM) with the (Clus-DoC) algorithm that combines cluster detection and colocalization analysis to observe the organization of 5-lipoxygenase (5-LO) and 5-lipoxygenase–activating protein (FLAP) into higher order assemblies on the nuclear envelope of mast cells; these assemblies were linked to leukotriene (LT) C4 production. In this study we investigated whether higher order assemblies of 5-LO and FLAP included cytosolic phospholipase A2 (cPLA2) and were linked to LTB4 production in murine neutrophils. Using two- and three-color dSTORM supported by fluorescence lifetime imaging microscopy we identified higher order assemblies containing 40 molecules (median) (IQR: 23, 87) of 5-LO, and 53 molecules (62, 156) of FLAP monomer. 98 (18, 154) molecules of cPLA2 were clustered with 5-LO, and 77 (33, 114) molecules of cPLA2 were associated with FLAP. These assemblies were tightly linked to LTB4 formation. The activation-dependent close associations of cPLA2, FLAP, and 5-LO in higher order assemblies on the nuclear envelope support a model in which arachidonic acid is generated by cPLA2 in apposition to FLAP, facilitating its transfer to 5-LO to initiate LT synthesis.

We previously reported that overexpression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) increases lung cancer cell proliferation by activating RAS signaling and that CYP24A1 knockdown inhibits tumor growth. However, the mechanism of CYP24A1-mediated cancer cell proliferation remains unclear. Here, we conducted cell synchronization and biochemical experiments in lung adenocarcinoma cells, revealing a link between CYP24A1 and anaphase-promoting complex (APC), a key cell cycle regulator. We demonstrate that CYP24A1 expression is cell cycle–dependent; it was higher in the G2-M phase and diminished upon G1 entry. CYP24A1 has a functional destruction box (D-box) motif that allows binding with two APC adaptors, CDC20-homologue 1 (CDH1) and cell division cycle 20 (CDC20). Unlike other APC substrates, however, CYP24A1 acted as a pseudo-substrate, inhibiting CDH1 activity and promoting mitotic progression. Conversely, overexpression of a CYP24A1 D-box mutant compromised CDH1 binding, allowing CDH1 hyperactivation, thereby hastening degradation of its substrates cyclin B1 and CDC20, and accumulation of the CDC20 substrate p21, prolonging mitotic exit. These activities also occurred with a CYP24A1 isoform 2 lacking the catalytic cysteine (Cys-462), suggesting that CYP24A1's oncogenic potential is independent of its catalytic activity. CYP24A1 degradation reduced clonogenic survival of mutant KRAS-driven lung cancer cells, and calcitriol treatment increased CYP24A1 levels and tumor burden in Lsl-KRASG12D mice. These results disclose a catalytic activity-independent growth-promoting role of CYP24A1 in mutant KRAS-driven lung cancer. This suggests that CYP24A1 could be therapeutically targeted in lung cancers in which its expression is high.

Endorepellin, the C-terminal fragment of the heparan sulfate proteoglycan perlecan, influences various signaling pathways in endothelial cells by binding to VEGFR2. In this study, we discovered that soluble endorepellin activates the canonical stress signaling pathway consisting of PERK, eIF2α, ATF4, and GADD45α. Specifically, endorepellin evoked transient activation of VEGFR2, which, in turn, phosphorylated PERK at Thr980. Subsequently, PERK phosphorylated eIF2α at Ser51, upregulating its downstream effector proteins ATF4 and GADD45α. RNAi-mediated knockdown of PERK or eIF2α abrogated the endorepellin-mediated up-regulation of GADD45α, the ultimate effector protein of this stress signaling cascade. To functionally validate these findings, we utilized an ex vivo model of angiogenesis. Exposure of the aortic rings embedded in 3D fibrillar collagen to recombinant endorepellin for 2–4 h activated PERK and induced GADD45α vis à vis vehicle-treated counterparts. Similar effects were obtained with the established cellular stress inducer tunicamycin. Notably, chronic exposure of aortic rings to endorepellin for 7–9 days markedly suppressed vessel sprouting, an angiostatic effect that was rescued by blocking PERK kinase activity. Our findings unravel a mechanism by which an extracellular matrix protein evokes stress signaling in endothelial cells, which leads to angiostasis.

Sperm head shaping is a key event in spermiogenesis and is tightly controlled via the acrosome–manchette network. Linker of nucleoskeleton and cytoskeleton (LINC) complexes consist of Sad1 and UNC84 domain–containing (SUN) and Klarsicht/ANC-1/Syne-1 homology (KASH) domain proteins and form conserved nuclear envelope bridges implicated in transducing mechanical forces from the manchette to sculpt sperm nuclei into a hook-like shape. However, the role of LINC complexes in sperm head shaping is still poorly understood. Here we assessed the role of SUN3, a testis-specific LINC component harboring a conserved SUN domain, in spermiogenesis. We show that CRISPR/Cas9-generated Sun3 knockout male mice are infertile, displaying drastically reduced sperm counts and a globozoospermia-like phenotype, including a missing, mislocalized, or fragmented acrosome, as well as multiple defects in sperm flagella. Further examination revealed that the sperm head abnormalities are apparent at step 9 and that the sperm nuclei fail to elongate because of the absence of manchette microtubules and perinuclear rings. These observations indicate that Sun3 deletion likely impairs the ability of the LINC complex to transduce the cytoskeletal force to the nuclear envelope, required for sperm head elongation. We also found that SUN3 interacts with SUN4 in mouse testes and that the level of SUN4 proteins is drastically reduced in Sun3-null mice. Altogether, our results indicate that SUN3 is essential for sperm head shaping and male fertility, providing molecular clues regarding the underlying pathology of the globozoospermia-like phenotype.

Myostatin (or growth/differentiation factor 8 (GDF8)) is a member of the transforming growth factor β superfamily of growth factors and negatively regulates skeletal muscle growth. Its dysregulation is implicated in muscle wasting diseases. SRK-015 is a clinical-stage mAb that prevents extracellular proteolytic activation of pro- and latent myostatin. Here we used integrated structural and biochemical approaches to elucidate the molecular mechanism of antibody-mediated neutralization of pro-myostatin activation. The crystal structure of pro-myostatin in complex with 29H4-16 Fab, a high-affinity variant of SRK-015, at 2.79 Å resolution revealed that the antibody binds to a conformational epitope in the arm region of the prodomain distant from the proteolytic cleavage sites. This epitope is highly sequence-divergent, having only limited similarity to other closely related members of the transforming growth factor β superfamily. Hydrogen/deuterium exchange MS experiments indicated that antibody binding induces conformational changes in pro- and latent myostatin that span the arm region, the loops contiguous to the protease cleavage sites, and the latency-associated structural elements. Moreover, negative-stain EM with full-length antibodies disclosed a stable, ring-like antigen–antibody structure in which the two Fab arms of a single antibody occupy the two arm regions of the prodomain in the pro- and latent myostatin homodimers, suggesting a 1:1 (antibody:myostatin homodimer) binding stoichiometry. These results suggest that SRK-015 binding stabilizes the latent conformation and limits the accessibility of protease cleavage sites within the prodomain. These findings shed light on approaches that specifically block the extracellular activation of growth factors by targeting their precursor forms.

Lethal infections by strains of the highly-pathogenic avian influenza virus (HPAIV) H5N1 pose serious threats to both the poultry industry and public health worldwide. A lack of confirmed HPAIV epitopes recognized by cytotoxic T lymphocytes (CTLs) has hindered the utilization of CD8+ T-cell–mediated immunity and has precluded the development of effectively diversified epitope-based vaccination approaches. In particular, an HPAIV H5N1 CTL-recognized epitope based on the peptide MHC-I–β2m (pMHC-I) complex has not yet been designed. Here, screening a collection of selected peptides of several HPAIV strains against a specific pathogen-free pMHC-I (pBF2*1501), we identified a highly-conserved HPAIV H5N1 CTL epitope, named HPAIV–PA123–130. We determined the structure of the BF2*1501–PA123–130 complex at 2.1 Å resolution to elucidate the molecular mechanisms of a preferential presentation of the highly-conserved PA123–130 epitope in the chicken B15 lineage. Conformational characteristics of the PA123–130 epitope with a protruding Tyr-7 residue indicated that this epitope has great potential to be recognized by specific TCRs. Moreover, significantly increased numbers of CD8+ T cells specific for the HPAIV–PA123–130 epitope in peptide-immunized chickens indicated that a repertoire of CD8+ T cells can specifically respond to this epitope. We anticipate that the identification and structural characterization of the PA123–130 epitope reported here could enable further studies of CTL immunity against HPAIV H5N1. Such studies may aid in the development of vaccine development strategies using well-conserved internal viral antigens in chickens.

The C-type lectin receptors (CLRs) form a family of pattern recognition receptors that recognize numerous pathogens, such as bacteria and fungi, and trigger innate immune responses. The extracellular carbohydrate-recognition domain (CRD) of CLRs forms a globular structure that can coordinate a Ca2+ ion, allowing receptor interactions with sugar-containing ligands. Although well-conserved, the CRD fold can also display differences that directly affect the specificity of the receptors for their ligands. Here, we report crystal structures at 1.8–2.3 Å resolutions of the CRD of murine dendritic cell-immunoactivating receptor (DCAR, or Clec4b1), the CLR that binds phosphoglycolipids such as acylated phosphatidyl-myo-inositol mannosides (AcPIMs) of mycobacteria. Using mutagenesis analysis, we identified critical residues, Ala136 and Gln198, on the surface surrounding the ligand-binding site of DCAR, as well as an atypical Ca2+-binding motif (Glu-Pro-Ser/EPS168–170). By chemically synthesizing a water-soluble ligand analog, inositol-monophosphate dimannose (IPM2), we confirmed the direct interaction of DCAR with the polar moiety of AcPIMs by biolayer interferometry and co-crystallization approaches. We also observed a hydrophobic groove extending from the ligand-binding site that is in a suitable position to interact with the lipid portion of whole AcPIMs. These results suggest that the hydroxyl group-binding ability and hydrophobic groove of DCAR mediate its specific binding to pathogen-derived phosphoglycolipids such as mycobacterial AcPIMs.

Lens proteins become increasingly cross-linked through nondisulfide linkages during aging and cataract formation. One mechanism that has been implicated in this cross-linking is glycation through formation of advanced glycation end products (AGEs). Here, we found an age-associated increase in stiffness in human lenses that was directly correlated with levels of protein–cross-linking AGEs. α-Crystallin in the lens binds to other proteins and prevents their denaturation and aggregation through its chaperone-like activity. Using a FRET-based assay, we examined the stability of the αA-crystallin–γD-crystallin complex for up to 12 days and observed that this complex is stable in PBS and upon incubation with human lens–epithelial cell lysate or lens homogenate. Addition of 2 mm ATP to the lysate or homogenate did not decrease the stability of the complex. We also generated complexes of human αA-crystallin or αB-crystallin with alcohol dehydrogenase or citrate synthase by applying thermal stress. Upon glycation under physiological conditions, the chaperone–client complexes underwent greater extents of cross-linking than did uncomplexed protein mixtures. LC-MS/MS analyses revealed that the levels of cross-linking AGEs were significantly higher in the glycated chaperone–client complexes than in glycated but uncomplexed protein mixtures. Mouse lenses subjected to thermal stress followed by glycation lost resilience more extensively than lenses subjected to thermal stress or glycation alone, and this loss was accompanied by higher protein cross-linking and higher cross-linking AGE levels. These results uncover a protein cross-linking mechanism in the lens and suggest that AGE-mediated cross-linking of α-crystallin–client complexes could contribute to lens aging and presbyopia.

Antibodies are widely used as cancer therapeutics, but their current use is limited by the low number of antigens restricted to cancer cells. A receptor tyrosine kinase, receptor tyrosine kinase-like orphan receptor 2 (ROR2), is normally expressed only during embryogenesis and is tightly down-regulated in postnatal healthy tissues. However, it is up-regulated in a diverse set of hematologic and solid malignancies, thus ROR2 represents a candidate antigen for antibody-based cancer therapy. Here we describe the affinity maturation and humanization of a rabbit mAb that binds human and mouse ROR2 but not human ROR1 or other human cell-surface antigens. Co-crystallization of the parental rabbit mAb in complex with the human ROR2 kringle domain (hROR2-Kr) guided affinity maturation by heavy-chain complementarity-determining region 3 (HCDR3)-focused mutagenesis and selection. The affinity-matured rabbit mAb was then humanized by complementarity-determining region (CDR) grafting and framework fine tuning and again co-crystallized with hROR2-Kr. We show that the affinity-matured and humanized mAb retains strong affinity and specificity to ROR2 and, following conversion to a T cell–engaging bispecific antibody, has potent cytotoxicity toward ROR2-expressing cells. We anticipate that this humanized affinity-matured mAb will find application for antibody-based cancer therapy of ROR2-expressing neoplasms.

Type IV filaments (T4F), which are helical assemblies of type IV pilins, constitute a superfamily of filamentous nanomachines virtually ubiquitous in prokaryotes that mediate a wide variety of functions. The competence (Com) pilus is a widespread T4F, mediating DNA uptake (the first step in natural transformation) in bacteria with one membrane (monoderms), an important mechanism of horizontal gene transfer. Here, we report the results of genomic, phylogenetic, and structural analyses of ComGC, the major pilin subunit of Com pili. By performing a global comparative analysis, we show that Com pili genes are virtually ubiquitous in Bacilli, a major monoderm class of Firmicutes. This also revealed that ComGC displays extensive sequence conservation, defining a monophyletic group among type IV pilins. We further report ComGC solution structures from two naturally competent human pathogens, Streptococcus sanguinis (ComGCSS) and Streptococcus pneumoniae (ComGCSP), revealing that this pilin displays extensive structural conservation. Strikingly, ComGCSS and ComGCSP exhibit a novel type IV pilin fold that is purely helical. Results from homology modeling analyses suggest that the unusual structure of ComGC is compatible with helical filament assembly. Because ComGC displays such a widespread distribution, these results have implications for hundreds of monoderm species.

Barbara H. Braffett
May 1, 2020; 69:1000-1010
Complications

Andrew S. Kimball
Sep 1, 2017; 66:2459-2471
Immunology and Transplantation

Mandeep Bajaj
Nov 1, 2005; 54:3148-3153
Metabolism

Maria Sörhede Winzell
Dec 1, 2004; 53:S215-S219
Section V: The Incretin Pathway

Lena Eliasson
May 1, 2020; 69:804-812
Small Noncoding RNAs in Diabetes

Anne Jörns
Apr 1, 2020; 69:624-633
Islet Studies

Marc Y. Donath
Dec 1, 2005; 54:S108-S113
Section III: Inflammation and beta-Cell Death

Ranjit Unnikrishnan
Jun 1, 2017; 66:1432-1442
Perspectives in Diabetes

Mikael Knip
Dec 1, 2005; 54:S125-S136
Section IV: Polygenic Disease and Environment

Roy Taylor
Apr 1, 2012; 61:778-779
Commentary

Mario Luca Morieri
Apr 1, 2020; 69:771-783
Genetics/Genomes/Proteomics/Metabolomics

Aldi T. Kraja
Apr 1, 2011; 60:1329-1339
Genetics

Edwin A.M. Gale
Dec 1, 2002; 51:3353-3361
Perspectives in Diabetes

Mary C. Gannon
Sep 1, 2004; 53:2375-2382
Pathophysiology

Miriam Cnop
Dec 1, 2005; 54:S97-S107
Section III: Inflammation and beta-Cell Death

Bruce B. Duncan
Jul 1, 2003; 52:1799-1805
Pathophysiology

Ralph A. DeFronzo
Apr 1, 2009; 58:773-795
Banting Lecture

Cheng Hu
Jan 1, 2018; 67:3-11
Perspectives in Diabetes

Frank Robinson's tenure as Giants manager was short but significant. He and his teams provided hope and promise when both were in short supply around Candlestick Park.

Buster Posey plans to be on the field when Giants pitchers and catchers begin their first workout at Scottsdale Stadium on Wednesday, and if his rehab from right hip surgery continues to go well, he expects the same for Opening Day.

Giants president of baseball operations Farhan Zaidi would prefer to stay mum when it comes to his forays into the free-agent market, but he realized there was no use in attempting to obscure the club's meeting with superstar Bryce Harper in Las Vegas earlier this week.

Madison Bumgarner has made it known he's not a fan of the "opener," a strategy of utilizing a pitcher -- usually a reliever -- to get the first few outs of a game before bringing in a pitcher who would usually start or pitch long relief.

With Spring Training set to kick off Tuesday, it feels like an opportune time to put together a way-too-early look at who might be with the Giants when they begin their regular season against the Padres on March 28.

One year after making a pair of high-profile acquisitions in Andrew McCutchen and Evan Longoria, the Giants have experienced a far slower and quieter winter, leaving the club with quite a few question marks as pitchers and catchers reported to Scottsdale, Ariz., for the start of Spring Training on Tuesday.

The 30 prospects below all are getting very long looks this spring with an eye toward breaking camp with the parent club. Even if they start the year in the Minors, they all should get the chance to contribute at some point in the very near future.

Who do the Giants have in the pipeline? Get scouting reports, video, stats, projected ETAs and more for San Francisco's Top 30 Prospects on MLB Pipeline's Prospect Watch.

Heading into his 13th year in the Majors, Cameron Maybin brings a fresh approach at the plate and a revamped swing as he looks to carve out playing time in the Giants' outfield.

1 November 2019

The 'Security and Prosperity in Asia' conference looked at the impact of international law in the Asia-Pacific with a focus on regional economic and security issues such as the South China Sea disputes.

6 November 2019 , Volume 95, Number 6

3 December 2019