ELVs released from adipose tissue.
Others have demonstrated previously that exosomes are released by 3T3-L1, a precursor adipocyte cell line (29
). We found that more ELVs are released in 30-min ex vivo adipose tissue cultures of age-matched wild-type (B6) mice fed a HFD over 3 months (HFDELVs, 16.5 ± 1.2 μg/g of adipose weight) and of leptin-deficient (ob/ob
) B6 mice (obELVs, 14.4 ± 1.1 μg/g of adipose weight) when compared to adipose tissue cultures of wild-type lean B6 mice (wtELVs, 4.1 ± 1.0 μg/g of adipose weight). The quantity of ELVs released from adipose tissue increased over a period from 30 min to 6 h of ex vivo adipose tissue culture (supplemental Fig. S1A
), that is, 16.5–22.2 μg/g HFDELVs; 14.4– 23.2 μg/g obELVs; and 4.1–6.7 μg/g wtELVs. Electron microscopy examination revealed vesicles that measured ~60–100 nm in diameter and had a cup-shaped morphology (supplemental Fig. S1B
). Neither calnexin nor Lamp-1 were detectable when the ELVs were analyzed by immunoblotting (supplemental Fig. S1C
), indicating that our ELV preparations were free of contaminating nonexosome membrane proteins (31
). Further evidence that the vesicles were exosomes was obtained through analysis of the protein composition using linear ion trap mass spectrometery (LTQ LC/MS) (supplemental Table S1). These analyses indicated a protein composition typical of exosomes derived from other cell types (32
) (supplemental Table S2). Specific proteins were at undetectable levels in wt-ELVs when compared with protein detected in obELVs and HFDELVs (supplemental Table S1), but in both cases the ELVs contained proteins known to be involved in cell metabolism, membrane trafficking, multiple small GTP-binding proteins, integral membrane proteins, and several class E vacuolar protein sorting proteins. The fatty acid composition of the obELVs was almost exclusively palmitic acid (n
= 8, 40.22 ± 3.82%) and stearic acid (52.71 ± 4.42%) (supplemental Table S3). Because there are minor differences in the protein composition of ELVs released from B6 mice fed an HFD in comparison with obELVs (supplemental Table S1), we focused on the B6 obELVs released from adipose tissues at 30-min ex vivo culture for the remainder of study because a larger amount of adipose tissue for isolation of ELVs was available from ob/ob
mice of the same age.
ELVs released from the adipose tissue of ob/ob mice activate monocytes.
We determine whether obELVs released from adipose tissue are taken up by cells of the immune system. We labeled obELVs with PKH67 dye before injecting them intravenously into B6 mice fed an HFD or standard diet for 3 months starting at 2 months of age. FACS analysis of single-cell suspensions of several tissues that had been harvested 24 h after the injection of obELVs indicated that more than 1.1% of total blood cells or liver leukocytes had taken up obELVs. Approximately 80% of the cells having taken up obELVs were CD11bF4/80+ monocytes (supplemental Fig. S2A and B), but no B cells, T cells, nor natural killer cells had taken up obELVs (data not shown). This result is unlikely because of PKH67 dye leakage from obELVs to the monocytes as no PKH67- positive macrophages were detected in the tissues of mice having been injected intravenously with free PKH67 dye (data not shown). FACS analysis of the gated PKH67+ cells isolated from peripheral blood further indicated that the monocytes (CD11b+F4/80+PKH67+) that had taken up the obELVs expressed higher levels of the monocyte receptors ICAM-1 (intracellular adhesion molecule-1), CD204, and MHCII (major histocompatibility complex II) than did monocytes that had taken up ELVs isolated from lean, wild-type B6 mice (wtELVs) (A), although both wtELVs and obELVs were taken up by the monocytes with equal efficiency (data not shown). In addition, at days 1 and 7 after injection of the ELVs there were higher levels of the proinflammatory cytokines IL-6 and TNF-α in the sera of the mice that had been injected with obELVs than in the sera of B6 mice that had been injected with wtELVs (B). Analysis of ELISA results indicated that the injection of ELVs does not induce the host to generate antibodies against the injected ELVs (data not shown), implying that induction of inflammatory cytokines may not lead to further activation of adaptive immune responses. The experiments as described above were also repeated in B6 mice fed a standard rat diet, and similar results were obtained (data not shown), suggesting that HFD preconditioning is not required for obELV-mediated activation of monocytes.
FIG. 1. Adipose obELVs activate macrophages. A: Wild-type B6 mice fed an HFD for 3 months starting at 2 months of age were injected intravenously with the PKH67+-labeled obELVs or wtELVs (30 μg/mouse). Twenty-four hours after the injection, CD11b+F4/80 (more ...)
ObELV-mediated activation of macrophages impairs glucose uptake and the insulin response of myocytes in vitro.
To determine whether the obELV-activated macrophages produce factors that affect insulin sensitivity, we determined the effect of conditioned medium harvested from bone marrow precursor cells that had been pretreated with obELVs for 14 days on glucose uptake and the insulin response of myocytes. The conditioned medium was harvested from 14-day cultures of bone marrow cells that had been pretreated with obELVs or wtELVs and cultured in the presence or absence of GM-CSF. In the course of these experiments, we noted that on day 4 after the addition of the ELVs to the day 0 cultured bone marrow precursor cells that the bone marrow precursors exhibited differentiation into macrophages instead of dendritic cells and that the differentiation into macrophages occurred when the dendritic cell differentiation factor GM-CSF had been added to the day 0 cultures (supplemental Fig. S3A). This differentiation did not occur when wtELVs (supplemental Fig. S3A) or thymus exosomes (supplemental Fig. S3D) were added to the day 0 bone marrow precursor cultures. These obELV-stimulated macrophages continued to proliferate until day 14 (supplemental Fig. S3B), even in the absence of growth factors. These activated macrophages secreted higher quantities of macrophage colony-stimulating factor, IL-6, and TNF-α into the culture supernatants than did the bone marrow precursors that had been stimulated with wtELVs (supplemental Fig. S3C) or thymus exosomes (supplemental Fig. S3E). Furthermore, elevated obELV concentrations were associated with the promotion of bone marrow precursor differentiation (supplemental Fig. S4A), proliferation (supplemental Fig. S4B), and induction of IL-6 (supplemental Fig. S4C).
Upon addition of the conditioned medium to the myocyte cultures, the levels of phosphorylation of Akt were lower in the myocytes cultured with the conditioned medium harvested from macrophages pretreated with obELVs than in the myocytes cultured with conditioned medium from wtELVs (A). Furthermore, basal and insulin-stimulated transport of glucose was inhibited in myocytes treated with obELV-conditioned medium, indicating that insulin function is also impaired (B). The conditioned medium–induced impairment of insulin responses was not observed when bone marrow precursors were stimulated with thymus exosomes (B), suggesting that obELV-mediated impairment of insulin responses in myocytes is adipose tissue exosome specific.
FIG. 2. Adipose obELVs promote the differentiation and proliferation of BMDMs and impair activation of the insulin signaling pathway in vitro and macrophage infiltration into adipose tissues in vivo. C2C12 cells at 80% confluency were cultured for 24 h in the (more ...)
In addition, in agreement with the data published by other groups (36
), addition of anti–TNF-α and anti–IL-6 neutralizing antibodies to the conditioned medium harvested from the obELV-treated wild-type bone marrow precursor cells led to a partial reversal of the impaired responses (supplemental Fig. S5).
To assess the homing of the macrophages that had taken up the ELVs, we injected B6 mice fed an HFD over 3 months with a mixture of PKH26-labeled BMDMs that had been preincubated with obELVs (BMDMsPKH26+) with PKH67- labeled BMDMs that had been preincubated with wtELVs (BMDMsPKH67+). FACS analysis of the tissues harvested 14 days after the BMDMsPKH26+ or BMDMsPKH67+ injections revealed that the number of PKH26+ macrophages were remarkably higher in adipose tissue and liver but not in the spleen and bone marrow (C). Analysis of the proliferation of the infiltrating macrophages (CD11b+F4/80+PKH26+) using a BrdU incorporation assay suggested that the macrophages that had been pretreated with obELVs proliferated faster than those pretreated with wtELVs (D). Preferential homing to and faster proliferation of macrophages prepulsed with obELVs, but not wtELVs, in adipose and liver tissue was also observed in the same-aged B6 ob/ob mice (data not shown).
ObELV-induced activation of macrophages is dependent on the TLR4 pathway.
The TLR pathway has been shown to play a role in the development of obesity (14
); therefore, we repeated the above studies using BMDMs from TLR2 knockout, TLR4 knockout, and B6 wild-type mice. ELISA analysis of supernatants obtained from the obELV-stimulated BMDMs show there were much higher levels of IL-6 and TNF-α in the culture medium of the obELV-treated BMDMs from TLR2 knockout and wild-type B6 mice than the obELV-treated BMDMs from TLR4 knockout mice (A
). FACS analysis of the cells indicated that the obELV-induced expression of CD204 was also lower in the obELV-treated BMDMs from the TLR4 knockout mice than the obELV-treated BMDMs from the wild-type B6 mice and the TLR2 knockout mice (supplemental Fig. S6). Similar results were obtained when ICAM and MHCII expression were analyzed by FACS (data not shown). Together, these data suggest that obELVs are capable of utilizing TLR4 signaling to induce a macrophage inflammatory response. To further substantiate the involvement of the TLR4 signaling pathway, we sought to determine if knockout of either MyD88 or TRIF affected the response, as both these molecules can be used as adaptors for the TLR4 signaling pathway (39
). Knockout of TRIF, but not MyD88, substantially inhibited the obELV-mediated induction of IL-6 and TNF-α (B
), suggesting that this response is TRIF dependent.
FIG. 3. The TLR4/TRIF pathway plays a role in adipose obELV-mediated macrophage activation and the impairment of the insulin response. A: BMDMs from wild-type B6, TLR2 knockout, or TLR4 knockout mice were treated with obELVs (10 μg/ml), and the quantity (more ...)
In addition, the combined medium harvested at 24 h after addition of obELV (10 μg/ml) to BMDMs from TLR4 knockout B6 mice led to better glucose uptake or insulin response of myocytes than from wild-type B6 mice (C).
ObELV RBP4 plays a role in the adipose obELV-mediated, TLR4-dependent induction of macrophage IL-6 and TNF-α.
RBP4 knockout mice display enhanced insulin sensitivity (18
). We found that RBP4 is present in ELVs isolated from the adipose tissue of B6 ob
mice and in significantly higher amounts than wtELVs (A
) isolated from age-matched mice. The addition of recombinant RBP4 (rRBP4) alone to BMDMs from wild-type B6 mice induced the production of IL-6 and TNF-α in a concentration-dependent manner (supplemental Fig. S7A
). This RBP4-induced production of IL-6 and TNF-α was dependent on TLR4 because there was less induction of either cytokine when TLR4 knockout macrophages were used in experiments (B
). This result was also supported by in vivo data indicating that 6 h after intravenous injection of recombinant RBP4 (250 μg/mouse) in TLR4 knockout B6 mice, a reduced induction of serum TNF-α and IL-6 had occurred (supplemental Fig. S7B
). Nuclear factor (NF)-κB activation has been shown to play a role in TLR4-driven inflammatory responses. This RBP4-induced production of IL-6 and TNF-α was also attenuated when BMDMs from NF-κB p50 knockout mice were used (supplemental Fig. S7C
). Collectively, these data suggest that RBP4-mediated induction of TNF-α and IL-6 is regulated through TLR4/NF-κB pathway.
FIG. 4. ObELV RBP4 induces the production of macrophage proinflammatory cytokines via activation of the TLR4/TRIF pathway. A: Fifty micrograms of obELVs and wtELVs were lysed in protein lysis buffer, and each lysate was resolved by PAGE in a 10% SDS gel for Western (more ...)
To address the possibility that ELV RBP4 utilizes a different pathway to stimulate TNF-α and IL-6 production by BMDMs than does free RBP4, we used a competitive assay. The BMDMs were pretreated 24 h before the addition of obELVs with mouse RBP4 or its dialyzed buffer as a control. ELISA analysis of the supernatants confirmed that obELVs or RBP4 alone could induce the production of TNF-α and IL-6; however, pretreatment of the BMDMs with RBP4 attenuated the ability of the adipose obELVs to induce the production of TNF-α and IL-6 (C), suggesting that RBP4 may compete with the same pathway as obELVs to induce TNF-α and IL-6.
ELVs from the adipose tissue of ob/ob mice induce insulin resistance in mice.
To test the effects of ELVs in vivo, B6 and TLR4 knockout mice were injected intravenously (30 μg/mouse) every 3 days for 3 weeks with ELVs released from the adipose tissue of lean wild-type mice or ob/ob mice. Glucose uptake, insulin response, and serum levels of TNF-α and IL-6 were determined after the last injection. Remarkably, the injection of ELVs released from the adipose tissue of ob/ob mice, but not wild-type mice, led to the development of glucose intolerance (A) and insulin resistance in wild-type mice (B). Moreover, the levels of glucose intolerance (A), insulin resistance (B), and serum TNF-α and IL-6 (C) were much lower in the TLR4 knockout mice that were treated with the obELVs than the wtELVs. Thus, ELVs released from the adipose tissue of ob/ob mice substantially enhance the development of insulin resistance and impair glucose tolerance and induction of inflammatory cytokines in a TLR4-dependent manner.
FIG. 5. Injection of obELVs leads to the intolerance of glucose uptake, insulin resistance, and induction of inflammatory cytokines of mice. A and B: Wild-type B6 mice or TLR4 knockout of B6 mice (n = 10) were injected intravenously with obELVs or wtELVs (30 (more ...)