Interleukin-1β Regulates Fat-Liver Crosstalk in Obesity by Auto-Paracrine Modulation of Adipose Tissue Inflammation and Expandability

The inflammasome has been recently implicated in obesity-associated dys-metabolism. However, of its products, the specific role of IL-1β was clinically demonstrated to mediate only the pancreatic beta-cell demise, and in mice mainly the intra-hepatic manifestations of obesity. Yet, it remains largely unknown if IL-1β, a cytokine believed to mainly function locally, could regulate dysfunctional inter-organ crosstalk in obesity. Here we show that High-fat-fed (HFF) mice exhibited a preferential increase of IL-1β in portal compared to systemic blood. Moreover, portally-drained mesenteric fat transplantation from IL-1βKO donors resulted in lower pyruvate-glucose flux compared to mice receiving wild-type (WT) transplant. These results raised a putative endocrine function for visceral fat-derived IL-1β in regulating hepatic gluconeogenic flux. IL-1βKO mice on HFF exhibited only a minor or no increase in adipose expression of pro-inflammatory genes (including macrophage M1 markers), Mac2-positive crown-like structures and CD11b-F4/80-double-positive macrophages, all of which were markedly increased in WT-HFF mice. Further consistent with autocrine/paracrine functions of IL-1β within adipose tissue, adipose tissue macrophage lipid content was increased in WT-HFF mice, but significantly less in IL-1βKO mice. Ex-vivo, adipose explants co-cultured with primary hepatocytes from WT or IL-1-receptor (IL-1RI)-KO mice suggested only a minor direct effect of adipose-derived IL-1β on hepatocyte insulin resistance. Importantly, although IL-1βKOs gained weight similarly to WT-HFF, they had larger fat depots with similar degree of adipocyte hypertrophy. Furthermore, adipogenesis genes and markers (pparg, cepba, fabp4, glut4) that were decreased by HFF in WT, were paradoxically elevated in IL-1βKO-HFF mice. These local alterations in adipose tissue inflammation and expansion correlated with a lower liver size, less hepatic steatosis, and preserved insulin sensitivity. Collectively, we demonstrate that by promoting adipose inflammation and limiting fat tissue expandability, IL-1β supports ectopic fat accumulation in hepatocytes and adipose-tissue macrophages, contributing to impaired fat-liver crosstalk in nutritional obesity.     

Niche-Dependent Gene Expression Profile of Intratumoral Heterogeneous Ovarian Cancer Stem Cell Populations

Intratumoral heterogeneity challenges existing paradigms for anti-cancer therapy. We have previously demonstrated that the human embryonic stem cells (hESC)-derived cellular microenvironment in immunocompromised mice, enables functional distinction of heterogeneous tumor cells, including cells which do not grow into a tumor in a conventional direct tumor xenograft platform. We have identified and characterized six cancer cell subpopulations each clonally expanded from a single cell, derived from human ovarian clear cell carcinoma of a single tumor, to demonstrate striking intratumoral phenotypic heterogeneity that is dynamically dependent on the tumor growth microenvironment. These cancer cell subpopulations, characterized as cancer stem cell subpopulations, faithfully recapitulate the full spectrum of histological phenotypic heterogeneity known for human ovarian clear cell carcinoma. Each of the six subpopulations displays a different level of morphologic and tumorigenic differentiation wherein growth in the hESC-derived microenvironment favors growth of CD44+/aldehyde dehydrogenase positive pockets of self-renewing cells that sustain tumor growth through a process of tumorigenic differentiation into CD44-/aldehyde dehydrogenase negative derivatives. Strikingly, these derivative cells display microenvironment-dependent plasticity with the capacity to restore self-renewal markers and CD44 expression. In the current study, we delineate the distinct gene expression and epigenetic profiles of two such subpopulations, representing extremes of phenotypic heterogeneity in terms of niche-dependent self-renewal and tumorigenic differentiation. By combining Gene Set Enrichment, Gene Ontology and Pathway-focused array analyses with methylation status, we propose a suite of robust differences in tumor self-renewal and differentiation pathways that underlie the striking intratumoral phenotypic heterogeneity which characterize this and other solid tumor malignancies.     

CpG Islands as a putative source for animal miRNAs: evolutionary and functional implications

MicroRNAs (miRs) are considered major contributors to the evolution of animal morphological complexity. Multiple bursts of novel miR families were documented throughout animal evolution, yet, their evolutionary origins are not understood. Here, we discuss two alternative genomic sources for novel miR families, namely, transposable elements, which were previously described, and a newly proposed origin: CpG islands. We show that these two origins are evolutionarily distinct and that they correspond to marked differences in several functional and genomic characteristics. Together, our results shed light on the intriguing origin of one of the major constituents of regulatory networks in animals, miRs.     

MDC1 is ubiquitylated on its tandem BRCT domain and directly binds RAP80 in a UBC13-dependent manner

The cellular response to DNA damage is essential for maintenance of genomic stability. MDC1 is a key member of the DNA damage response. It is an adaptor protein that binds and recruits proteins to sites of DNA damage, a crucial step for a proper response. MDC1 contains several protein-protein interacting modules, including a tandem BRCT domain that mediates various interactions involving MDC1. Here we demonstrate that MDC1 binds directly to RAP80, which is a DNA damage response protein that recruits BRCA1 to sites of damage. The interaction between MDC1 and RAP80 requires the tandem BRCT domain of MDC1 and the ubiquitin-interacting motifs of RAP80. Moreover, the interaction depends on UBC13, an E2 ubiquitin ligase that catalyzes K63-linked poly-ubiquitin chain formation. The results highly propose that the interaction between MDC1 and RAP80 depends on a ubiquitylation event, which we found to take place on K-1977 of MDC1. This study provides the first evidence that interactions involving MDC1 can be regulated by ubiquitylation.     

A comparative assessment of fluo Ca2+ indicators in rat ventricular myocytes

The fluo family of indicators is frequently used in studying Ca(2+) physiology; however, choosing which fluo indicator to use is not obvious. Indicator properties are typically determined in well-defined aqueous solutions. Inside cells, however, the properties can change markedly. We have characterized each of three fluo variants (fluo-2MA, fluo-3 and fluo-4) in two forms-the acetoxymethyl (AM) ester and the K(+) salt. We loaded indicators into rat ventricular myocytes and used confocal microscopy to monitor depolarization-induced fluorescence changes and fractional shortening. Myocytes loaded with the indicator AM esters showed significantly different Ca(2+) transients and fractional shortening kinetics. Loading the K(+) salts via whole-cell patch-pipette eliminated differences between fluo-3 and fluo-4, but not fluo-2MA. Cells loaded with different indicator AM esters showed different staining patterns-suggesting differential loading into organelles. Ca(2+) dissociation constants (K(d,Ca)), measured in protein-rich buffers mimicking the cytosol were significantly higher than values determined in simple buffers. This increase in K(d,Ca) (decrease in Ca(2+) affinity) was greatest for fluo-3 and fluo-4, and least for fluo-2MA. We conclude that the structurally-similar fluo variants differ with respect to cellular loading, subcellular compartmentalization, and intracellular Ca(2+) affinity. Therefore, judicious choice of fluo indicator and loading procedure is advisable when designing experiments.     

Spatiotemporal dynamics of re-innervation and hyperinnervation patterns by uninjured CGRP fibers in the rat foot sole epidermis after nerve injury

Background

Minocycline prevents the development of neuropathic and inflammatory pain by inhibiting microglial activation and postsynaptic currents. But, how minocycline obviates acute visceral pain is unclear. The present study investigated whether minocycline had an any antinociceptive effect on acetic acid-induced acute abdominal pain after intraperitoneal (i.p.) administration of saline or minocycline 1 hour before acetic acid injection (1.0%, 250 ??l, i.p.).

Results

Minocycline (4, 10, or 40 mg/kg) significantly decreased acetic acid-induced nociception (0-60 minutes post-injection) and the enhancement in the number of c-Fos positive cells in the T5-L2 spinal cord induced by acetic acid injection. Also, the expression of spinal phosphorylated extracellular signal-regulated kinase (p-ERK) induced by acetic acid was reduced by minocycline pre-administration. Interestingly, intrathecal introduction of PD98059, an ERK upstream kinase inhibitor, markedly blocked the acetic acid-stimulated pain responses.

Conclusions

These results demonstrate that minocycline effectively inhibits acetic acid-induced acute abdominal nociception via the inhibition of neuronal p-ERK expression in the spinal cord, and that minocycline may have therapeutic potential in suppressing acute abdominal pain.     

Spatiotemporal dynamics of re-innervation and hyperinnervation patterns by uninjured CGRP fibers in the rat foot sole epidermis after nerve injury

ABSTRACT: The epidermis is innervated by fine nerve endings that are important in mediating nociceptive stimuli. However, their precise role in neuropathic pain is still controversial. Here, we have studied the role of epidermal peptidergic nociceptive fibers that are located adjacent to injured fibers in a rat model of neuropathic pain. Using the Spared Nerve Injury (SNI) model, which involves complete transections of the tibial and common peroneal nerve while sparing the sural and saphenous branches, mechanical hypersensitivity was induced of the uninjured lateral (sural) and medial (saphenous) area of the foot sole. At different time points, a complete foot sole biopsy was taken from the injured paw and processed for Calcitonin Gene-Related Peptide (CGRP) immunohistochemistry. Subsequently, a novel 2D-reconstruction model depicting the density of CGRP fibers was made to evaluate the course of denervation and re-innervation by uninjured CGRP fibers. The results show an increased density of uninjured CGRP-IR epidermal fibers on the lateral and medial side after a SNI procedure at 5 and 10 weeks. Furthermore, although in control animals the density of epidermal CGRP-IR fibers in the footpads was lower compared to the surrounding skin of the foot, 10 weeks after the SNI procedure, the initially denervated footpads displayed a hyper-innervation. These data support the idea that uninjured fibers may play a considerable role in development and maintenance of neuropathic pain and that it is important to take larger biopsies to test the relationship between innervation of injured and uninjured nerve areas.     

Mistic: Cellular localization,solution behavior,polymerization, and fibril formation

Mistic represents a family of unique membrane‐associating proteins originally found in Bacillus subtilis (M110). As a fusion partner, it has been shown to assist overexpression of foreign integral membrane proteins in E. coli. We have expressed shorter Mistic homologs from other Bacillus species and surprisingly, unlike M110, found them abundant in the cytoplasm. These Mistic homologs including the corresponding shorter sequence (amino acids 27 through 110 of M110) exist as multimeric assemblies in solution in the absence of detergent. Crystals of Mistic from B. leicheniformis (M2) diffracted to 3.2 Å resolution, indicating that it exists as a multimer in the crystalline state as well. Moreover, we show that although M2 is mostly α‐helical, it tends to polymerize and form fibrils. Such oligomerization could potentially mask the charged surface of the monomeric Mistic to assist membrane integration.