Adipose-derived circulating miRNAs regulate gene expression in other tissues

Abstract

Adipose tissue is a major site of energy storage and has a role in the regulation of metabolism through the release of adipokines. Here we show that mice with an adipose-tissue-specific knockout of the microRNA (miRNA)-processing enzyme Dicer (ADicerKO), as well as humans with lipodystrophy, exhibit a substantial decrease in levels of circulating exosomal miRNAs. Transplantation of both white and brown adipose tissue-brown especially-into ADicerKO mice restores the level of numerous circulating miRNAs that are associated with an improvement in glucose tolerance and a reduction in hepatic Fgf21 mRNA and circulating FGF21. This gene regulation can be mimicked by the administration of normal, but not ADicerKO, serum exosomes. Expression of a human-specific miRNA in the brown adipose tissue of one mouse in vivo can also regulate its 3' UTR reporter in the liver of another mouse through serum exosomal transfer. Thus, adipose tissue constitutes an important source of circulating exosomal miRNAs, which can regulate gene expression in distant tissues and thereby serve as a previously undescribed form of adipokine.

Extended Data Figure 1

(a) Electron microscopy of exosomes isolated from ADicerKO serum by differential centrifugation. (b) EXOCET ELISA assay measuring CETP protein in exosome samples, corresponding to isolated exosome number from serum of ADicerKO (KO) or littermate mice (Lox). (c) qNano assay measuring exosome numbers and size based on Tunable Resistive Pulse Sensing technology (IZON) from exosome preparations from ADicerKO or Lox mice. (d) Principle Component Analysis of exosomal miRNA levels in ADicerKO (KO) and Lox (WT), n=4 per group. Error bars represent SEM.

Extended Data Figure 2

(a) Heatmap showing Z-scores of miRNA expression measurements from whole serum ADicerKO (KO) or littermate wild type mice (WT) and exosomal miRNAs from ADicerKO (KO) or littermate wild type mice (WT) (n=4 per group). (b) Heatmap showing Z-scores of miRNA expression measurements of exosomal miRNAs from culture supernatant of Dicer^fl/fl preadipocytes transduced with Ad-GFP (GFP) or Ad-CRE(CRE) (n=3 per group). (c) Heatmap showing Z-scores of miRNA expression measurements of exosomal miRNAs from serum of 4-week old ADicerKO (ADicerKO) and Lox (Control) mice (n=3 per group).

Extended Data Figure 3

(a) Demographic information of human patients with HIV lipodystrophy (HIV), congenital generalized lipodystrophy (CGL) or normal subjects. (b) EXOCET ELISA assay measuring CETP protein as a measure of exosome number from isolated from human sera of individuals with HIV Lipodystrophy, congenital generalized lipodystrophy (CGL) and normal subjects (n=4 per group). (c) Principle Component Analysis of exosomal miRNA expression in HIV Lipodystrophy, CGL, and control subjects, n=4 per group. Error bars represent SEM.

Extended Data Figure 4

(a) Principle Component Analysis of miRNA expression in mouse fat depots: epididymal (Epi), inguinal (Ing), and brown adipose tissue (BAT), n=4 per group. (b) Weights of the transplanted epididymal (Epi), inguinal (Ing), and brown adipose tissue (BAT) at time of transplantation into ADicerKO mice (white bars) and at time of sacrifice (black bars), n=3 (c) Weights of ADicerKO mice undergoing sham surgery (SAL) or with transplanted epididymal (Epi), inguinal (Ing), or brown adipose tissue (BAT) and Lox (WT) mice. Error bars represent SEM.

Extended Data Figure 5

(a) Principle Component Analysis of serum exosomal miRNA levels in ADicerKO after sham surgery (Sham) or transplantation with inguinal fat (ING), with epididymal fat (EPI) or BAT, and Lox controls (WT), n=4 per group. (b) Circulating insulin and adipokine levels in WT, ADicerKO, or transplanted ADicerKO mice (n=3 per group, two-tailed t-test, p<0.05).

Extended Data Figure 6

(a) FGF21 mRNA levels as assessed by qPCR in liver (LIV), BAT, inguinal (Ing), epididymal (Epi), pancreas (Panc), kidney (Kidn), and quadriceps muscle (Quad) of ADicerKO (black bars) or Lox (white bars) (n=4 per group, p=0.0286, two-tailed Mann Whitney U test). (b) Relative abundance (log2FC) as assessed by qPCR of miR-99a, miR-99b, and miR-100 in exosomes from ADicerKO undergoing fat transplantation surgery compared to sham, n=4 per group.

Extended Data Figure 7

(a) FGF21 3’UTR luciferase activity in murine liver cells (AML-12) following introduction of miR-99a, miR-99b, miR-100 or miR-466i (10 nM of miRNA mimic) by direct electroporation (n=3 per group, p=0.003, two-tailed t-test). (b) FGF21 mRNA abundance in murine liver cells (AML-12) following transduction with miRNA mimics of miR-99a, miR-99b, miR-100 or miR-466i (10 nM) (n=3 per group, p=0.037, two tailed t-test). (c) Hepatic FGF21 mRNA levels by qPCR followed by 48 hrs incubation of AML-12 hepatic cells with exosomes derived from ADicerKO or Lox littermates (WT) mice or with ADicerKO-isolated exosomes electroporated with 10nM of miR-99a, miR-99b, miR-100 or miR-466i (n=3 per group, p=0.0001, two-tailed t-test). Error bars represent SEM.

Extended Data Figure 8

(a) qPCR of mature miR-16, miR-201, and miR-222 in liver of Lox mice (WT), ADicerKO mice (KO), and ADicerKO transplanted with BAT (KO+BAT) (n=3 per group, p=0.02 for miR-16, p=0.002 for miR-201, and p=0.028 for miR-222; one-way Analysis of variance. Significant comparisons were identified by Tukey’s multiple comparisons test). (b) qPCR of pre-miR-16, pre-miR-201, and pre-miR-222 abundance in liver of Lox mice (WT), ADicerKO mice (KO), and ADicerKO transplanted with BAT (KO+BAT) (n=3 per group, p<0.05, one-way analysis of variance). (c) CT values of qPCR of Adenoviral DNA isolated from BAT-p1 and liver-p1 (experimental protocol 1) and liver-p2 (experimental protocol 2) detecting Adenoviral LacZ or pre-miR-302f (n=4 per group). Error bars represent SEM.

Figure 1. Fat tissue is a major source of exosomal miRNAs

(a) Schematic showing creation of ADicerKO mice. (b) Immunoelectron microscopy of CD63 and CD9 in murine serum exosomes. (c) Heatmap showing Z-scores of exosomal miRNAs from serum of ADicerKO (KO) and Lox (WT) mice (n=4/group). (d) Waterfall plot showing relative abundance of serum exosomal miRNAs between ADicerKO and control mice (n=4/group, p<0.05). (e) Heatmap showing Z-scores of exosomal miRNAs in sera of humans with HIV lipodystrophy (HIV), congenital generalized lipodystrophy (CGL) and controls (n=4/group). (f) Waterfall plots representing the relative abundance of exosomal miRNAs differentially expressed between HIV lipodystrophy, generalized lipodystrophy and controls (n=4/group, p<0.05). (g) Venn diagrams representing significantly up- and down-regulated miRNAs in HIV and CGL compared to controls (n=4/group, p<0.05).

Figure 2. Fat depot contributions to circulating exosomal miRNAs

(a) Schematic of fat transplantation experiment using WT donor fat depots transplanted into ADicerKO recipients. (b) Heatmap showing Z-scores of miRNA expression in inguinal (Ing), epididymal (Epi), and brown adipose tissue (BAT) from WT donor mice (n=4/group). Venn diagram represents number of fat depot-specific miRNAs with expression >U6 in WT mice (n=4/group). (c) Heatmap of Z-scores of exosomal miRNAs in ADicerKO or C57Bl/6 mice after sham surgery and transplantation of fat (n=4/group). The Venn diagram represents miRNAs reconstituted at least 50% of the way to WT values after transplantation (n=4/group, p<0.05). (d) Glucose tolerance test in C57Bl/6 and AdicerKO mice (n=3/group, p=0.0001, WT vs KO at 0 min; p=0.013, WT vs KO at 15 min; p=0.0001, WT vs KO at 90 min, two-tailed t-test). (e) Area under the curve (AUC) of glucose tolerance tests in ADicerKO (+sham surgery), C57Bl/6 (+sham surgery) and ADicerKO mice after fat transplantion; (n=3/group, p=0.0002, WT vs KO, p=0.033 KO vs KO+BAT, two-tailed t-test). Bars represent SEM.

Figure 3. Fat-derived exosomal miRNAs regulate hepatic FGF21 and transcription

(a) Enzyme-linked immunoassay (ELISA) of FGF21 in serum of ADicerKO and control littermates (n=4/group, p=0.028, two-tailed Mann-Whitney U-test). (b) qPCR of hepatic FGF21 mRNA in Lox and ADicerKO mice (n=4/group, p=0.028, two-tailed Mann-Whitney U-test). (c) Serum FGF21 of ADicerKO (+sham surgery), WT mice (+sham surgery) and ADicerKO transplanted groups (n=3/group, p=0.019, WT vs KO+BAT, two-tailed t-test). (d) qPCR of hepatic FGF21 mRNA of mice in Panel c (n=3/group, p=0.046, Cont vs KO+BAT, two-tailed t-test). (e) FGF21-3’UTR luciferase activity after incubation of AML-12 cells with exosomes from Lox (exoWT), ADicerKO (exoKO), 10 nM free miR-99b or exosomes derived from ADicerKO mice electroporated with miR-99b (exoKO+miR-99b) (n=3/group, p=0.008, WT vs KO, p=0.008, KO vs. KO+99b, two-tailed t-test) (f) FGF21-3’UTR activity after incubation of AML-12 cells with exosomes from ADicerKO, Lox littermates, or ADicerKO mice and electroporated to introduce miR-99a, miR-99b, miR-100 or miR-466i. (n=3/group, p=0.0007, exoWT vs. exoKO, p=0.002, exoKO vs. exoKO+99b, two-tailed t-test).

Figure 4. In vivo regulation of FGF21 via exosomal miR-99b

(a) Lox (WT), ADicerKO (KO), and ADicerKO mice injected i.v. with wild-type exosomes (KO+exoWT) transduced with pacAd5-Luc-FGF21-3’UTR luciferase reporter and subjected to IVIS analysis. (b) Total flux luminescence by IVIS of above mice (n=3/group, p=0.039, Kruskal-Wallis ANOVA , WT vs KO, Dunn’s post-hoc test). (c) qPCR of hepatic FGF21 mRNA in above mice (n=3/group, p=0.039, Kruskal-Wallis ANOVA with Dunn’s post-hoc test). (d) ELISA of serum FGF21 of above mice (n=3/group, p=0.027, Kruskal-Wallis ANOVA with Dunn’s post-hoc test) (e) Lox mice injected i.v. with ADicerKO exosomes (WT+exoKO) and ADicerKO mice injected with either ADicerKO exosomes (KO+exoKO) or ADicerKO exosomes electroporated with miR-99b (KO+exomiR99b) subjected to IVIS analysis. (f) Total flux luminescence in IVIS from mice in Panel e. (n=3/group, p=0.079, Kruskal-Wallis ANOVA, Dunn’s post-hoc test). (g) qPCR of hepatic FGF21 mRNA of mice in Panel e (n=3 per group, p=0.039, Kruskal-Wallis ANOVA, significant comparison WT+exoKO vs KO+exoKO, Dunn’s post-hoc test). (h) ELISA of serum FGF21 of mice in Panel e. (n=3/group, p=0.027, Kruskal-Wallis ANOVA, significant comparison WT+exoKO vs KO+exoKO, Dunn’s post-hoc test). Error bars represent SEM.

Figure 5. BAT-derived exosomes expressing human miRNA miR-302f target their reporter in liver in vivo

(a) Protocol 1. Schematic of in vivo targeting protocol using adenovirus bearing pre-miR-302f or LacZ directly into BAT. (b) C57Bl/6 mice injected i.v. with pacAd5-hsa_miR-302f 3’-UTR reporter after BAT injection of Ad-pre-hsa-miR-302f or Ad-LacZ subjected to IVIS (n=4 per group). (c) Total flux luminescence obtained via IVIS analysis from mice in Panel B. (n=4/group, p=0.028, Mann-Whitney U-test). (d) Protocol 2. Schematic of in vivo targeting protocol injecting exosomes from C57Bl/6 mice transduced with pre-miR-302f or adenovirus bearing LacZ directly into BAT. (e) C57Bl/6 mice transduced with pacAd5-hsa_miR-302f 3’-UTR reporter after i.v. injections of serum exosomes from Ad-pre-hsa_miR-302f or Ad-LacZ BAT injected mice and subjected to IVIS analysis (n=4 per group). (f) Total flux luminescence obtained from using protocol in panel e (n=4/group, p=0.028, two-tailed Mann-Whitney U-test). Bars represent SEM. (g) Model of mechanisms by which fat-derived circulating exosomal miRNAs might regulate target mRNAs in other tissues.