JMJD8 is a Novel Molecular Nexus Between Adipocyte-Intrinsic Inflammation and Insulin Resistance

Chronic low-grade inflammation, often referred to as metainflammation, develops in response to overnutrition and is a major player in the regulation of insulin sensitivity. While many studies have investigated adipose tissue inflammation from the perspective of the immune cell compartment, little is known about how adipocytes intrinsically contribute to metainflammation and insulin resistance at the molecular level. Here, we demonstrate a novel role for Jumonji C Domain Containing Protein 8 (JMJD8) as an adipocyte-intrinsic molecular nexus between inflammation and insulin resistance. We determined that JMJD8 was highly enriched in white adipose tissue, especially in the adipocyte fraction. Adipose JMJD8 levels were dramatically increased in obesityassociated insulin resistance models. Its levels were increased by feeding and insulin, and inhibited by fasting. A JMJD8 gain-of-function was sufficient to drive insulin resistance, whereas loss-of-function improved insulin sensitivity in mouse and human adipocytes. Consistent with this, Jmjd8-ablated mice had increased whole-body and adipose insulin sensitivity and glucose tolerance on both chow and a high-fat diet, while adipocyte-specific Jmjd8-overexpressing mice displayed worsened whole-body metabolism on a high-fat diet. We found that JMJD8 affected the transcriptional regulation of inflammatory genes. In particular, it was required for LPS-mediated inflammation and insulin resistance in adipocytes. For this, JMJD8 required Interferon Regulatory Factor (IRF3) to mediate its actions in adipocytes. Together, our results demonstrate that JMJD8 acts as a novel molecular factor that drives adipocyte inflammation in conjunction with insulin sensitivity

Ebf1 suppresses <i>Zfp521</i> expression via an intronic binding site.

<p>(A) ChIP-Seq tracks corresponding to the Zfp521 locus are shown for three histone marks: H3K4me3 (green), H3K27Ac (black), and H3K36me3 (blue) at four time-points during 3T3-L1 adipogenesis. A cluster of Ebf1 peaks highlighted in the red box contains three putative Ebf1-responsive elements (EBF-RE). (B) ChIP-PCR analysis was performed with antibody against IgG or Ebf1 in 3T3-L1 preadipocytes. Immunoprecipitated DNA was amplified with Q-PCR using primers designed for the three putative EBF-REs shown in (A). (C) Zfp521 mRNA expression was measured in 3T3-L1 preadipocytes transduced with shScr or shEbf1. (D) Zfp521 mRNA expression was measured in confluent <i>Ebf1^+/+</i> and <i>Ebf1^−/−</i> MEFs. (E) Ebf1 and Zfp521 mRNA expression was measured in <i>Ebf1^+/+</i> and <i>Ebf1^−/−</i> MEFs transduced with Ebf1 or empty vector. Data presented as mean ± SD, <i>n</i> = 3, *<i>p</i><0.05. (F) A proposed model for the transcriptional cascade involving Zfp521, Ebf1, and Zfp423.</p

Zfp521 is a suppressor of adipogenesis in vitro.

<p>(A) C3H10T1/2 cells were differentiated and RNA isolated at the indicated time points. Gene expression of <i>Zfp521</i> and <i>Pparg</i> was measured by Q-PCR and normalized to cyclophilin. Data shown as mean of three biological replicates. (B) Protein lysates isolated during 3T3-L1 adipogenesis were subjected to western blotting with anti-Zfp521 antibody. (C, left) Zfp521 mRNA expression was measured in fractionated subcutaneous and epididymal fat tissue taken from wild-type mice (SV, stromal-vascular fraction; AD, adipocytes). (C, right) SV of epididymal fat tissue from Zfp423^GFP transgenic mice was sorted with GFP antibody and plated. After washing away floating cells, Zfp521 mRNA expression was measured in GFP− and GFP+ cells. (D–G) C3H10T1/2 cells were retrovirally transduced with Zfp521, empty vector, shRNA specific forZfp521 (sh521), or a scrambled hairpin (Scr). Overexpression and knock-down were confirmed by immunoblotting of Zfp521 prior to differentiation in the boxed insert. Cells were differentiated with DMI or DMI plus rosiglitazone (DMIR) and stained with oil red-O (D, F) and adipocyte markers were determined by Q-PCR (E, G) on day 8. Data presented as mean ± SD, <i>n</i> = 3, *<i>p</i><0.05.</p

Zfp521 suppresses Zfp423 expression.

<p>(A) 3T3-L1 preadipocytes were transduced with retrovirus expressing sh521, shScr, Zfp521, or empty vector. After puromycin selection, RNA was collected and submitted for analysis using Affymetrix arrays. The Venn diagram shows the number of genes up-regulated by sh521 (sh521/shScr>1.5-fold) and down-regulated by Zfp521 (Zfp521/pMSCV<0.7-fold). The heat map corresponds to genes in the intersecting set. (B) Overexpression of Zfp521 in C3H10T1/2 cells represses Zfp423 expression by Q-PCR. (C) Knockdown of Zfp521 in C3H10T1/2 cells enhances Zfp423 expression by Q-PCR. Data presented as mean ± SD, <i>n</i> = 3, *<i>p</i><0.05.</p

Reduction of Zfp521 enhances adipogenic potential in vivo.

<p>(A) Sections of the subcutaneous region of e18.5 <i>Zfp521^+/+</i> and <i>Zfp521^−/−</i> embryos were stained with anti-FABP4 (red) and DAPI (blue). The magnified section shows representative FABP4⁺, lipid-filled cells. (B) ∼20–25 images per embryo were used to quantify the number of lipid-filled and FABP4-positive adipocytes. <i>Zfp521^+/+</i>, <i>n</i> = 4 embryos; <i>Zfp521^−/−</i>, <i>n</i> = 6 embryos; *<i>p</i><0.05. (C, D) Representative histology (HE staining) of the interscapular BAT region from <i>Zfp521^+/+</i> and <i>Zfp521^−/−</i> e18.5 embryos (red arrowhead, interscapular BAT; white arrow, skeletal muscle) (C) and BAT and liver weight relative to total body weight (D). Data presented as mean ± SD, <i>Zfp521^+/+</i> (<i>n</i> = 9); <i>Zfp521^−/−</i> (<i>n</i> = 15), *<i>p</i><0.05. (E) shZfp521 and scrambled hairpin (shScr) expressing F442A cells were mixed and injected into nude mice. Percentage contribution of shScr and sh521-expressing cells in fat pads determined using specific Q-PCR (see text for details). Data presented as mean ± SD, <i>n</i> = 11, *<i>p</i><0.05.</p

Zfp521 inhibits Ebf1 transcriptional activity through physical interaction.

<p>(A) 3T3-L1 preadipocytes were harvested and endogenous Ebf1 was immunoprecipitated using anti-Ebf1 beads and blotted with normal goat-IgG or anti-Zfp521. (B–D) 3T3-L1 preadipocytes were co-transfected with vectors expressing Flag-Zfp521, Myc-Ebf1, and various reporter plasmids containing the <i>mb1</i>-promoter (B), <i>Sncg</i>-promoter (C), or <i>Cebpa</i>-promoter (D). At 24 h after transfection, luciferase activity was normalized to β-galactosidase activity. Data presented as mean ± SD, <i>n</i> = 4, *<i>p</i><0.05. (E) 3T3-L1 preadipocytes were stably transduced with Flag-Ebf1 or Flag-Zfp521. ChIP assay was performed on 3T3-L1 cells that were treated with DMI for 1 h using anti-Flag antibody or an IgG control using PCR primers directed at regions of the Cebpa containing the putative Ebf sites. (F, G) Immortalized <i>Zfp521^+/+</i> and <i>Zfp521^−/−</i> MEFs were transduced with a retrovirus expressing Ebf1, Zfp521, or empty vector and differentiated prior to staining with oil red-O after 8 d (F) and adipocyte gene expression was measured by Q-PCR (G). (H, I) C3H10T1/2 cells were transduced with a retrovirus expressing Zfp521WT, Zfp521ΔZF27-30, Zfp521Δ13aa, or empty pMSCV vector. Cells were differentiated with DMIR and stained with oil red-O and gene expression was measured on day 6. (J) Zfp521 and Ebf1 were expressed in C3H10T1/2 cells alone or in combination, and expression of Zfp521, Ebf1, and Zfp423 was determined by Q-PCR. (K) Zfp521, Zfp521Δ27-30, or pMSCV was expressed in C3H10T1/2 cells and expression of Zfp521, Ebf1, and Zfp423 was determined by Q-PCR. Data presented as mean ± SD, <i>n</i> = 3, *<i>p</i><0.05.</p

AIFM2 is required for high-intensity aerobic exercise by promoting glucose utilization

   Skeletal muscle is a major regulator of glycemic control at rest and glucose utilization increases drastically during exercise. Sustaining a high glucose utilization via glycolysis requires efficient replenishment of NAD+ in the cytosol. Apoptosis-inducing mitochondrion-associated factor 2 (AIFM2) has previously been shown to be a NADH oxidoreductase domain–containing flavoprotein to promote glycolysis for diet and cold-induced thermogenesis. Here, we find that AIFM2 is selectively and highly induced in glycolytic extensor digitorum longus (EDL) muscle during exercise. Overexpression of AIFM2 in myotubes is sufficient to elevate the NAD+/NADH ratio, increasing the glycolytic rate. Thus, overexpression of AIFM2 in skeletal muscle greatly increases exercise capacity, with increased glucose utilization. Conversely, muscle-specific Aifm2 depletion via in vivo transfection of hairpins against Aifm2 or tamoxifen-inducible haploinsufficiency of Aifm2 in muscles decreases exercise capacity and glucose utilization in mice. Moreover, muscle-specific introduction of NDE1, Saccharomyces cerevisiae external NADH dehydrogenase, NDE, ameliorates impairment in glucose utilization and exercise intolerance of the muscle-specific Aifm2 haploinsufficient mice. Together, we show a novel role for AIFM2 as a critical metabolic regulator for efficient utilization of glucose in glycolytic EDL muscles. </p

Computer simulation data.

<p>EPBD based Langevin molecular dynamics showing the intrinsic breathing dynamics in the promoter regions of PPARG and PPARA genes: vertical axis - size of bubbles [base pairs, bp] with defined average lifetimes; horizontal axis - nucleotide position [base pair] where the TSS is labeled ‘+1’. The color bar on the right indicates the bubble lifetimes in pico seconds [ps]. The promoter sequences obtained from the DBTSS (<a href="http://dbtss.hgc.jp" target="_blank">http://dbtss.hgc.jp</a>), are shown above the simulation data panel. The red letter indicates the TSS position. The identity of the gene sequence is shown above the plots.</p

Gene specific effect of THz irradiation in mouse stem cell cultures.

<p>a) Gene expression profiling of MSC cells in response to 9 hours of THz exposure. Of ∼21,000 annotated genes represented in the Affymetrix mouse genome microarray, 1050 were underexpressed (red) and 1154 were overexpressed (green) with statistical significance p<0.05 as compared to the non-irradiated parallel control; b) RT-PCR measurement for selected genes. The relative level of gene expression in response to 9 hours exposure of MSC to THz radiation normalized to the TBP gene. The identity of the genes is shown below the bars. Cells without THz treatment served as control. The experimental results are consistent between three independent RT-PCR measurements in duplicates and in two different sets of irradiation. Brown bars – THz irradiated cells; blue bars – control cells. The table lists the S-Scores (representing the change in expression level in response to THz irradiation compared to the nonirradiated control) for these genes derived from the Affymetrix mouse genome microarray. c) The temperature was monitored using an IR detector, and separately using thermo-sensors glued to the outside of the petri dish lids. The temperature at the end of irradiation for the control plate with cells (c-79.4⁰F) and the THz irradiated cells (THz-79.9⁰F) is shown above the snap shots panels (snap shots are from the IR detectors in Fahrenheit). d) RT-PCR measurements for selected genes in response to 2, 4, and 9 hours irradiation. RNA levels are normalized to the TBP gene. The identity of the gene is shown at the top. The duration of irradiation is shown below the bars in hours (h). The number of specific transcripts is shown on the vertical axis in relative units [R.U.]. The RT-PCR results are consistent between two independent sets of measurements in duplicates.</p

THz irradiation of mouse stem cells.

<p>a) THz radiation was generated by a frequency-doubling BBO crystal, in Argon at 600 torr pressure, with 1 KHz repetition rate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015806#pone.0015806-Kim1" target="_blank">[31]</a>. The irradiated and control cells were in thermal contact and the temperature of both samples was monitored by themosensors. b) Mouse stem cells were monitored by light microscopy for morphological changes in response to THz exposure. A significant accumulation of lipid-like droplets in the cellular cytoplasm was visible in response to 6 hours of THz irradiation. Representative photographs are shown with 100x magnification for: Cells without THz exposure; cells after 2 hours of THz exposure; and cells after 6 hours THz exposure. Cells with an increased number of lipid-like droplets inclusions in the cytosol are indicated with black arrow; orange arrows – undifferentiated cells; white arrows – initial stage of adipogenesis.</p