Molecular and Metabolic Analysis of Arsenic-Exposed Humanized AS3MT Mice.

BACKGROUND: Chronic exposure to inorganic arsenic (iAs) has been associated with type 2 diabetes (T2D). However, potential sex divergence and the underlying mechanisms remain understudied. iAs is not metabolized uniformly across species, which is a limitation of typical exposure studies in rodent models. The development of a new "humanized" mouse model overcomes this limitation. In this study, we leveraged this model to study sex differences in the context of iAs exposure. OBJECTIVES: The aim of this study was to determine if males and females exhibit different liver and adipose molecular profiles and metabolic phenotypes in the context of iAs exposure. METHODS: Our study was performed on wild-type (WT) 129S6/SvEvTac and humanized arsenic +3 methyl transferase (human AS3MT) 129S6/SvEvTac mice treated with 400 ppb of iAs via drinking water ad libitum. After 1 month, mice were sacrificed and the liver and gonadal adipose depots were harvested for iAs quantification and sequencing-based microRNA and gene expression analysis. Serum blood was collected for fasting blood glucose, fasting plasma insulin, and homeostatic model assessment for insulin resistance (HOMA-IR). RESULTS: We detected sex divergence in liver and adipose markers of diabetes (e.g., miR-34a, insulin signaling pathways, fasting blood glucose, fasting plasma insulin, and HOMA-IR) only in humanized (not WT) mice. In humanized female mice, numerous genes that promote insulin sensitivity and glucose tolerance in both the liver and adipose are elevated compared to humanized male mice. We also identified Klf11 as a putative master regulator of the sex divergence in gene expression in humanized mice. DISCUSSION: Our study underscored the importance of future studies leveraging the humanized mouse model to study iAs-associated metabolic disease. The findings suggested that humanized males are at increased risk for metabolic dysfunction relative to humanized females in the context of iAs exposure. Future investigations should focus on the detailed mechanisms that underlie the sex divergence. https://doi.org/10.1289/EHP12785.

14-3-3-zeta mediates GLP-1 receptor agonist action to alter α cell proglucagon processing.

Recent studies demonstrate that α cells contribute to glucose-stimulated insulin secretion (GSIS). Glucagon-like peptide-1 receptor (GLP-1R) agonists potently potentiate GSIS, making these drugs useful for diabetes treatment. However, the role of α and ß cell paracrine interactions in the effects of GLP-1R agonists is undefined. We previously found that increased ß cell GLP-1R signaling activates α cell GLP-1 expression. Here, we characterized the bidirectional paracrine cross-talk by which α and ß cells communicate to mediate the effects of the GLP-1R agonist, liraglutide. We find that the effect of liraglutide to enhance GSIS is blunted by α cell ablation in male mice. Furthermore, the effect of ß cell GLP-1R signaling to activate α cell GLP-1 is mediated by a secreted protein factor that is regulated by the signaling protein, 14-3-3-zeta, in mouse and human islets. These data refine our understanding of GLP-1 pharmacology and identify 14-3-3-zeta as a potential target to enhance α cell GLP-1 production.

GLP-1 receptor signaling increases PCSK1 and ß cell features in human α cells.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that potentiates glucose-stimulated insulin secretion. GLP-1 is classically produced by gut L cells; however, under certain circumstances α cells can express the prohormone convertase required for proglucagon processing to GLP-1, prohormone convertase 1/3 (PC1/3), and can produce GLP-1. However, the mechanisms through which this occurs are poorly defined. Understanding the mechanisms by which α cell PC1/3 expression can be activated may reveal new targets for diabetes treatment. Here, we demonstrate that the GLP-1 receptor (GLP-1R) agonist, liraglutide, increased α cell GLP-1 expression in a ß cell GLP-1R-dependent manner. We demonstrate that this effect of liraglutide was translationally relevant in human islets through application of a new scRNA-seq technology, DART-Seq. We found that the effect of liraglutide to increase α cell PC1/3 mRNA expression occurred in a subcluster of α cells and was associated with increased expression of other ß cell-like genes, which we confirmed by IHC. Finally, we found that the effect of liraglutide to increase bihormonal insulin+ glucagon+ cells was mediated by the ß cell GLP-1R in mice. Together, our data validate a high-sensitivity method for scRNA-seq in human islets and identify a potentially novel GLP-1-mediated pathway regulating human α cell function.

Identification of an Antiretroviral Small Molecule That Appears To Be a Host-Targeting Inhibitor of HIV-1 Assembly.

Given the projected increase in multidrug-resistant HIV-1, there is an urgent need for development of antiretrovirals that act on virus life cycle stages not targeted by drugs currently in use. Host-targeting compounds are of particular interest because they can offer a high barrier to resistance. Here, we report identification of two related small molecules that inhibit HIV-1 late events, a part of the HIV-1 life cycle for which potent and specific inhibitors are lacking. This chemotype was discovered using cell-free protein synthesis and assembly systems that recapitulate intracellular host-catalyzed viral capsid assembly pathways. These compounds inhibit replication of HIV-1 in human T cell lines and peripheral blood mononuclear cells, and are effective against a primary isolate. They reduce virus production, likely by inhibiting a posttranslational step in HIV-1 Gag assembly. Notably, the compound colocalizes with HIV-1 Gag in situ; however, unexpectedly, selection experiments failed to identify compound-specific resistance mutations in gag or pol, even though known resistance mutations developed upon parallel nelfinavir selection. Thus, we hypothesized that instead of binding to Gag directly, these compounds localize to assembly intermediates, the intracellular multiprotein complexes containing Gag and host factors that form during immature HIV-1 capsid assembly. Indeed, imaging of infected cells shows compound colocalized with two host enzymes found in assembly intermediates, ABCE1 and DDX6, but not two host proteins found in other complexes. While the exact target and mechanism of action of this chemotype remain to be determined, our findings suggest that these compounds represent first-in-class, host-targeting inhibitors of intracellular events in HIV-1 assembly.IMPORTANCE The success of antiretroviral treatment for HIV-1 is at risk of being undermined by the growing problem of drug resistance. Thus, there is a need to identify antiretrovirals that act on viral life cycle stages not targeted by drugs in use, such as the events of HIV-1 Gag assembly. To address this gap, we developed a compound screen that recapitulates the intracellular events of HIV-1 assembly, including virus-host interactions that promote assembly. This effort led to the identification of a new chemotype that inhibits HIV-1 replication at nanomolar concentrations, likely by acting on assembly. This compound colocalized with Gag and two host enzymes that facilitate capsid assembly. However, resistance selection did not result in compound-specific mutations in gag, suggesting that the chemotype does not directly target Gag. We hypothesize that this chemotype represents a first-in-class inhibitor of virus production that acts by targeting a virus-host complex important for HIV-1 Gag assembly.

Identifying the assembly intermediate in which Gag first associates with unspliced HIV-1 RNA suggests a novel model for HIV-1 RNA packaging.

During immature capsid assembly, HIV-1 genome packaging is initiated when Gag first associates with unspliced HIV-1 RNA by a poorly understood process. Previously, we defined a pathway of sequential intracellular HIV-1 capsid assembly intermediates; here we sought to identify the intermediate in which HIV-1 Gag first associates with unspliced HIV-1 RNA. In provirus-expressing cells, unspliced HIV-1 RNA was not found in the soluble fraction of the cytosol, but instead was largely in complexes ≥30S. We did not detect unspliced HIV-1 RNA associated with Gag in the first assembly intermediate, which consists of soluble Gag. Instead, the earliest assembly intermediate in which we detected Gag associated with unspliced HIV-1 RNA was the second assembly intermediate (~80S intermediate), which is derived from a host RNA granule containing two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6. At steady-state, this RNA-granule-derived ~80S complex was the smallest assembly intermediate that contained Gag associated with unspliced viral RNA, regardless of whether lysates contained intact or disrupted ribosomes, or expressed WT or assembly-defective Gag. A similar complex was identified in HIV-1-infected T cells. RNA-granule-derived assembly intermediates were detected in situ as sites of Gag colocalization with ABCE1 and DDX6; moreover these granules were far more numerous and smaller than well-studied RNA granules termed P bodies. Finally, we identified two steps that lead to association of assembling Gag with unspliced HIV-1 RNA. Independent of viral-RNA-binding, Gag associates with a broad class of RNA granules that largely lacks unspliced viral RNA (step 1). If a viral-RNA-binding domain is present, Gag further localizes to a subset of these granules that contains unspliced viral RNA (step 2). Thus, our data raise the possibility that HIV-1 packaging is initiated not by soluble Gag, but by Gag targeted to a subset of host RNA granules containing unspliced HIV-1 RNA.

Formation of RNA Granule-Derived Capsid Assembly Intermediates Appears To Be Conserved between Human Immunodeficiency Virus Type 1 and the Nonprimate Lentivirus Feline Immunodeficiency Virus.

During immature capsid assembly in cells, human immunodeficiency virus type 1 (HIV-1) Gag co-opts a host RNA granule, forming a pathway of intracellular assembly intermediates containing host components, including two cellular facilitators of assembly, ABCE1 and DDX6. A similar assembly pathway has been observed for other primate lentiviruses. Here we asked whether feline immunodeficiency virus (FIV), a nonprimate lentivirus, also forms RNA granule-derived capsid assembly intermediates. First, we showed that the released FIV immature capsid and a large FIV Gag-containing intracellular complex are unstable during analysis, unlike for HIV-1. We identified harvest conditions, including in situ cross-linking, that overcame this problem, revealing a series of FIV Gag-containing complexes corresponding in size to HIV-1 assembly intermediates. Previously, we showed that assembly-defective HIV-1 Gag mutants are arrested at specific assembly intermediates; here we identified four assembly-defective FIV Gag mutants, including three not previously studied, and demonstrated that they appear to be arrested at the same intermediate as the cognate HIV-1 mutants. Further evidence that these FIV Gag-containing complexes correspond to assembly intermediates came from coimmunoprecipitations demonstrating that endogenous ABCE1 and the RNA granule protein DDX6 are associated with FIV Gag, as shown previously for HIV-1 Gag, but are not associated with a ribosomal protein, at steady state. Additionally, we showed that FIV Gag associates with another RNA granule protein, DCP2. Finally, we validated the FIV Gag-ABCE1 and FIV Gag-DCP2 interactions with proximity ligation assays demonstrating colocalization in situ Together, these data support a model in which primate and nonprimate lentiviruses form intracellular capsid assembly intermediates derived from nontranslating host RNA granules.IMPORTANCE Like HIV-1 Gag, FIV Gag assembles into immature capsids; however, it is not known whether FIV Gag progresses through a pathway of immature capsid assembly intermediates derived from host RNA granules, as shown for HIV-1 Gag. Here we showed that FIV Gag forms complexes that resemble HIV-1 capsid assembly intermediates in size and in their association with ABCE1 and DDX6, two host facilitators of HIV-1 immature capsid assembly that are found in HIV-1 assembly intermediates. Our studies also showed that known and novel assembly-defective FIV Gag mutants fail to progress past putative intermediates in a pattern resembling that observed for HIV-1 Gag mutants. Finally, we used imaging to demonstrate colocalization of FIV Gag with ABCE1 and with the RNA granule protein DCP2. Thus, we conclude that formation of assembly intermediates derived from host RNA granules is likely conserved between primate and nonprimate lentiviruses and could provide targets for future antiviral strategies.