Adipose-Derived Circulating miRNAs Regulate Gene Expression
in Other Tissues
Thomas Thomou
1
, Marcelo A. Mori
2
, Jonathan M. Dreyfuss
3,6
, Masahiro Konishi
1
, Masaji
Sakaguchi
1
, Christian Wolfrum
7
, Tata Nageswara Rao
1,8
, Jonathon N. Winnay
1
, Ruben
Garcia-Martin
1
, Steven K. Grinspoon
4
, Phillip Gorden
5
, and C. Ronald Kahn
1
1
Section on Integrative Physiology & Metabolism, Joslin Diabetes Center and Harvard Medical
School, Boston, MA
2
Department of Biochemistry and Tissue Biology, State University of
Campinas, Campinas, Brazil
3
Bioinformatics Core, Joslin Diabetes Center and Harvard Medical
School, Boston, MA
4
MGH Program in Nutritional Metabolism, Massachusetts General Hospital
and Harvard Medical School, Boston, MA
5
Diabetes, Endocrinology and Obesity Branch, NIDDK,
National Institutes of Health, Bethesda, MD
6
Department of Biomedical Engineering, Boston
University, Boston, MA
7
ETHZ, Department of Health Sciences and Metabolism, Zurich,
Switzerland
8
Department of Biomedicine, Experimental Hematology, University Hospital Basel,
Switzerland
Abstract
Adipose tissue is a major site of energy storage and plays a role in regulation of metabolism
through release of adipokines. Here we show that mice with a fat-specific knockout of the
miRNA-processing enzyme Dicer (ADicerKO), as well as humans with lipodystrophy, have major
decreases in circulating exosomal miRNAs. Transplantation of white and especially brown adipose
tissue (BAT) into ADicerKO mice restores circulating miRNAs associated with an improvement in
glucose tolerance and a reduction of hepatic FGF21 mRNA and circulating FGF21. This gene
regulation can be mimicked by administration of normal, but not AdicerKO, serum exosomes.
Expression of a human-specific miRNA in BAT of one mouse
in vivo
can also regulate its 3’UTR-
reporter in liver of another mouse through serum exosomal transfer. Thus, adipose tissue
constitutes a major source of circulating exosomal miRNAs, and these miRNAs can regulate gene
expression in distant tissues thereby serving as novel forms of adipokines.
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Corresponding Author: C. Ronald Kahn, MD, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215, Phone: (617) 309-2635,
c.ronald.kahn@joslin.harvard.edu.
The authors declare no competing financial interest.
Author Contributions
MAM assisted with experimental design, generated the ADicerKO mice and designed the Ad-Luc-FGF213’UTR constructs, JMD
carried out bioinformatics analysis, MK performed Adenoviral injections in BAT, MS assisted with retro-orbital injections, CW
created Ad-LacZ, Ad-pre-hsa-miR302f and Ad-Luc-miR302f-3’UTR Adenoviruses, TNT assisted with retroorbital and tail vain
injections, JNW assisted with fat depot miRNA PCR, RG-M assisted with IVIS experiments and in vitro luminescence assays, SKG
provided human HIV lipodystrophy sera samples, PG provided human CGL sera samples, TT and CRK designed the study, collected
and analyzed data, and wrote the manuscript.
HHS Public Access
Author manuscript
Nature
. Author manuscript; available in PMC 2017 August 15.
Published in final edited form as:
Nature
. 2017 February 23; 542(7642): 450–455. doi:10.1038/nature21365.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Keywords
Brown fat; Exosomes; Obesity; Lipodystrophy; Metabolic syndrome; FGF21; Tissue crosstalk
miRNAs are non-coding RNAs of 19–22 nucleotides that function as negative regulators of
translation and are involved in many cellular processes
1,2,3
. In addition to tissues, many
miRNAs exist in the circulation
4
, a large fraction of which are in exosomes
5
, i.e., 50–200nm
vesicles released from multivesicular bodies
6
. Increased levels of specific miRNAs have
been associated with a variety of diseases, including cancer
7
, diabetes
3,8,9
obesity
10
, and
cardiovascular disease
11
. miRNAs play an important role in the differentiation and function
of many cells, including adipose tissue
12
. We have shown that white adipose tissue (WAT)
miRNAs decline with age due to a decrease in the miRNA processing enzyme Dicer
13
and
are also reduced in humans with HIV-associated lipodystrophy
14
due to a decrease in Dicer.
To better understand the role of miRNAs in fat, we generated mice specifically lacking Dicer
in adipose tissue using Cre-lox gene recombination (Figure 1a)
13
. ADicerKO mice exhibit a
defect in miRNA processing in adipose tissue resulting in a reduction of WAT, whitening of
BAT, insulin resistance and altered circulating lipids
14
.
Adipose Tissue is a Major Source of Circulating Exosomal miRNAs
To determine to what extent adipose tissue contributes to circulating miRNAs, we isolated
exosomes from sera of 6-month-old male ADicerKO and control mice by differential
ultracentrifugation
15
. These vesicles were 80–200nm in diameter
16
(Extended Data Figure
1a) and stained for the exosomal markers CD63 and CD9 (Figure 1b)
17,18
. The number of
exosomes isolated from ADicerKO and controls was comparable (Extended Data Figure 1b
and 1c). qPCR profiling of serum exosomes for 709 murine miRNAs revealed 653
detectable miRNAs (defined as CT<34). Compared to control, ADicerKO mice exhibited
significant alterations in 422 exosomal miRNAs. Of these, 3 miRNAs were significantly
increased, while 419 had significant decreases (Figures 1c–d, Extended Data Figure 1d and
Supplemental Table 1) with 88% reduced by >4-fold, suggesting that adipose tissue is a
major source of circulating exosomal miRNAs. Consistent with this, many of the reduced
miRNAs (Supplemental Table 1) have been previously identified as highly expressed in fat,
including miR-221, miR-201, miR-222 and miR-16
9,19,20
. miRNAs also exist in the
circulation outside of exosomes. Indeed, in a sample of 80 miRNAs, there was a broad
reduction in total miRNAs in ADicerKO serum when compared to serum of WT mice
(Extended Data Figure 2a), however, this reduction was not as dramatic as the
downregulation of exosomal miRNAs showing that adipose contributes especially to the
exosomal miRNA fraction. The loss of exosomal miRNA secretion in adipocytes lacking
Dicer is cell autonomous. Thus, in preadipocytes isolated from Dicer-floxed animals and
recombined
in vitro
, most of the detectable miRNAs (of 380 miRNAs profiled) released in
exosomes into the media were decreased when compared to control Ad-GFP-transduced
cells (Extended Data Figure 2b).
To further dissociate altered metabolism from lipodystrophy as a cause of reduced exosomal
miRNAs, we compared serum miRNAs from 4-week-old control and AdicerKO mice, since
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at this age metabolic phenotypes of ADicerKO mice are minimal (Extended Data Figure 2c).
Again, of the 380 miRNAs profiled, 373 miRNAs were detectable with 202 down-regulated
in ADicerKO mice and only 23 miRNAs up-regulated, indicating that reduction in
circulating exosomal miRNAs reflects primarily differences in miRNA processing/
production rather than effects of chronic lipodystrophy.
To determine if circulating miRNAs in humans also originate from fat, we performed
exosomal miRNA profiling on sera from patients with congenital generalized lipodystrophy
(CGL) and patients with HIV-related lipodystrophy, previously shown to have decreased
levels of Dicer in adipose tissue
14
(Extended Data Figure 3a). Isolation yielded similar
exosome numbers from controls and lipodystrophic patients (Extended Data Figure 3b).
qPCR profiling of 572 miRNAs in exosomes revealed 119 significantly different between
control and HIV lipodystrophy subjects and 213 significantly different between control and
CGL subjects (Figures 1e–f, Extended Data Figure 3c, Supplemental Tables 2 and 3). Of
these, only 5% (29 miRNAs) were upregulated in CGL or HIV lipodystrophy, while 217
(38%) were down-regulated, with 75 decreased in both groups (Figure 1g, Supplemental
Table 4). Again, several of these miRNAs have been previously implicated in regulation of
fat
9,10,20,21
. Thirty miRNAs that were decreased in serum of both patient cohorts were also
decreased in the serum of ADicerKO mice (Supplemental Table 5).
Adipose Tissue Transplantation Reconstitutes Circulating miRNAs in
Lipodystrophic Mice
To verify that adipose tissue is indeed a major source of circulating miRNAs, we
transplanted fat from normal mice into ADicerKO mice (Figure 2a). miRNA profiling of
subcutaneous inguinal (Ing) WAT, intraabdominal epididymal (Epi) WAT, and BAT from the
normal donor mice revealed distinct, depot-specific signatures consistent with previous
studies
22
(Figures 2b, Extended Data Figure 4a; Supplemental Table 6). Considering only
miRNAs that were expressed greater than U6, 126 miRNAs were highly expressed in BAT,
106 in Ing-WAT, and 160 in Epi-WAT, with 82 in all three depots (Figure 2b). During the
following two weeks, all mice had maintained body weight, and at sacrifice the transplanted
fat weighed 80–90% of the original weight, indicating successful engraftment (Extended
Data Figures 4b and 4c). As in the first cohort, in sham-operated ADicerKO mice circulating
exosomal miRNAs were markedly reduced compared to controls (Figure 2c). By
comparison, ADicerKO mice that received fat transplants showed remarkable restoration of
circulating exosomal miRNAs (Figures 2c and Extended Data Figure 5a; Supplemental
Tables 7 and 8). Indeed, of the 177 circulating exosomal miRNAs that were detectable in
wild-type and significantly decreased in ADicerKO serum, fat transplantation restored the
levels of the majority of these at least 50% of the way to normal, indicating that adipose
tissue is a major source of circulating exosomal miRNAs and that different depots contribute
differentially.
Physiologically, ADicerKO mice had markedly impaired glucose tolerance tests (GTTs)
compared to controls with an ~50% increase in area under the curve (Figures 2d and 2e).
This showed only small changes after transplantation of Ing-WAT or Epi-WAT, however,
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GTT was significantly improved in the ADicerKO mice receiving BAT transplantation
(Figure 2e). ADicerKO mice also exhibit marked insulin resistance, as indicated by
increased circulating insulin levels; this was also reduced in the group receiving BAT
transplants, but did not quite reach statistical significance (Extended Data Figure 5b). Serum
IL-6, leptin and adiponectin levels were all lower in ADicerKO and were not restored by
transplantation (Extended Data Figure 5b).
FGF-21 as a Potential Target of Regulation by Circulating Exosomal
miRNAs
FGF21 (fibroblast growth factor-21) is produced in liver and other tissues, released into the
circulation and exerts effects on multiple tissues in control of metabolism
23
. ADicerKO mice
had a ~3-fold increase in circulating FGF21, associated with increased levels of FGF21
mRNA in liver, muscle, fat and pancreas (Figures 3a and 3b, Extended Data Figure 6a).
After transplantation of WAT, serum FGF21 and liver FGF21 mRNA remained unchanged in
the ADicerKO mice (Figures 3c and 3d). However, ADicerKO mice that received BAT
transplants showed an ~50% reduction in the FGF21 mRNA in liver (Figure 3d). This was
paralleled by a reduction of circulating FGF21 levels (Figure 3c), indicating that BAT
transplantation provided some factor(s) that directly or indirectly regulated FGF21
expression in liver. Considering that one factor could be circulating miRNAs, we performed
miRDB analysis to identify miRNAs that might target the 3’-UTR of murine FGF21
mRNA
24
. Four candidates were identified (miR-99a, miR-99b, miR-100, and miR-466i),
and three of these (miR-99a, −99b, and −100) were significantly decreased in the serum of
ADicerKO mice compared to controls. While these three miRNAs were restored to near WT
levels in all ADicerKO transplant groups, only ADicerKO mice receiving BAT transplant
exhibited expression levels higher than WT, tracking with reductions in circulating FGF21
levels in ADicerKO mice transplanted with BAT (Extended Data Figure 6b). To determine
which of these miRNAs might regulate FGF21, we transfected AML-12 liver cells with an
adenoviral pacAd5-FGF21 3’-UTR luciferase reporter and after 2 days transfected the cells
with 10 nM of a candidate or control miRNA mimetic. Of these, only miR-99b resulted in a
robust reduction of FGF21 luciferase activity (Extended Data Figure 7a), and this correlated
with a reduction in FGF21 mRNA level by 65% (Extended Data Figure 7b).
To test if these miRNAs could regulate FGF21 when presented in exosomes, we exposed
AML-12 cells expressing the FGF21-3’UTR luciferase reporter to exosomes from control or
ADicerKO mice or ADicerKO exosomes which had been electroporated with either
miR-99a, miR-99b, miR-100, miR-466i or a control mimic. We found that
in vitro
the
isolated exosomes from control mice were able to suppress FGF21-3’UTR luciferase
activity by 60%, whereas exosomes from ADicerKO serum had no effect (Figure 3e).
Furthermore, while ADicerKO exosomes reconstituted with miR-99a, miR-100 or miR-466i
had minimal effects, ADicerKO exosomes bearing miR-99b resulted in a ~55% suppression
of the luciferase activity (Figure 3f), and this was paralleled by an equal reduction in FGF21
mRNA levels, mimicking the effect of wild-type exosomes (Extended Data Figure 7c). This
regulation of FGF21 was dependent on exosomal delivery and was not recapitulated when
naked miR-99b was incubated with these cells (Figure 3e, right two bars)
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To address regulation of FGF21 by exosomal miRNAs
in vivo
, we transduced ADicerKO
and WT mice with a pacAd5-FGF21 3’-UTR luciferase reporter and measured hepatic
FGF21 suppression using the IVIS imaging system. Consistent with the
in vitro
study,
FGF21 3’-UTR activity
in vivo
was 5-fold higher in ADicerKO mice than WT mice,
reflecting the absence of repressive circulating miRNAs in the ADicerKO mice (Figures 4a
and 4b). Injection of WT-exosomes into AdicerKO mice induced suppression of the elevated
FGF21 reporter activity by ~60%. This was confirmed by qPCR which showed a reduction
in elevated hepatic FGF21 mRNA and a parallel decrease in circulating FGF21 compared to
KO mice (Figures 4c and 4d). Consistent with BAT-secreted exosome delivery of miRNAs to
liver, miRNAs miR-16, miR-201 and miR-222, which are relatively fat-specific, were
significantly decreased in livers of ADicerKO mice and restored toward normal by BAT
transplantation (Extended Data Figure 8a). This occurred with no change in the
corresponding pre-miRNA species in the liver (Extended Data Figure 8b).
In separate experiments, we injected WT and KO mice with KO exosomes with or without
reconstitution of miR-99b (Figure 4e). Again, KO mice showed 2.5-fold higher luciferase
activity than WT mice, when both were given KO exosomes. Administration of KO
exosomes reconstituted with miR-99b in the AdicerKO re-induced suppression of the
FGF21-3’-UTR reporter 45% of the way toward normal (Figure 4f). This was accompanied
by a parallel reduction in hepatic FGF21 message (Figure 4g) and reduced circulating
FGF21 (Figures 4h).
Regulation of Liver Gene Expression by Adipose-Produced Circulating
Exosomal miRNAs
Regulation of FGF21 is a complex process, which involves multiple factors. To define the
potential of adipose-derived circulating miRNAs
in vivo
, we developed a more specific
reporter system taking advantage of the human-specific miRNA hsa_miR-302f and its
3’UTR reporter
25
, since this miRNA does not have a mouse homolog. We then performed
two types of experiments. In the first protocol (Figure 5a) we injected adenovirus bearing
pre-hsa_miR-302f or its control directly into BAT to get BAT-specific expression of the
transduced gene
26
. Three days later, we injected the same mice intravenously (i.v.) with the
adenovirus 3’-UTR luciferase reporter for hsa_miR-302f to get its expression in liver. Only
if there was communication between the miRNA expressed in BAT and the reporter
expressed in liver would we observe suppression of the reporter. Indeed, IVIS analysis 5
days after transduction revealed that in mice with Ad-hsa_miR-302f transduced in BAT there
was a >95% reduction of luciferase activity in liver when compared to mice with LacZ-
control transduced into BAT (Figures 5b and 5c).
In protocol 2 (Figure 5d), to definitively address whether hsa_miR-302f suppression of its
reporter in liver was contingent on exosomal delivery, we used two separate cohorts of
C57Bl/6 mice. One cohort was transduced with adenovirus bearing pre-hsa_miR-302f or
control-LacZ adenovirus directly into BAT. A second, separate cohort of mice was
transduced in liver by i.v. injection of adenovirus bearing the 3’-UTR hsa_miR-302f
reporter. We then obtained serum from the donor cohorts over the following 8 days, isolated
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