Bone Marrow Mesenchymal Stem Cells-Derived Exosomal MiR-29b

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Bone Marrow Mesenchymal Stem Cells-Derived Exosomal MiR-29b-3p Regulates Aging-Associated Insulin Resistance Tian Su, Yuzhong Xiao, Ye Xiao, Qi Guo, Changjun Li, Yan Huang, Qiufang Deng, Jingxiang Wen, Fangliang Zhou, and Xiang-Hang Luo ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b09375 • Publication Date (Web): 04 Feb 2019 Downloaded from http://pubs.acs.org on February 5, 2019

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Identification and entry into the BMSCs

Intramedullary injection

miR-29b-3p

PEI-citrate core structure (nanocore) AntagomiR-29b-3p

Adipocytes

Aptamer

Myocytes

BMSCs

Hepatocytes

Exosomes with miR-29b-3p

Insulin sensitivity

Bone Marrow Mesenchymal Stem Cells-Derived Exosomal MiR-29b-3p Regulates Aging-Associated Insulin Resistance

ACS Paragon Plus Environment

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Bone Marrow Mesenchymal Stem Cells-Derived Exosomal MiR-29b-3p Regulates Aging-Associated Insulin Resistance

Tian Su#, 1Yuzhong Xiao#, 1Ye Xiao, 1Qi Guo, 1Changjun Li, 1Yan Huang, 1Qiufang

1

Deng, 1Jingxiang Wen, 2Fangliang Zhou, 1Xiang-Hang Luo*

Department of Endocrinology, Endocrinology Research Center, Xiangya Hospital of

1

Central South University, Changsha, Hunan, 410008, China; Department of Biochemistry and Molecular Biology, Hunan University of Chinese

2

Medicine, Changsha, Hunan, 410208, China;

Tian Su and Yuzhong Xiao were equally contributed to this work.

#

To whom correspondence should be addressed: Prof. Xiang-Hang Luo, Department of Endocrinology, Endocrinology Research Center, Xiangya Hospital of Central South University, 87# Xiangya Road, Changsha, Hunan 410008, China. Tel: +86-0731-89752728; Fax: +86-731-4327324; E-mail: [email protected];

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ABSTRACT Insulin resistance is the major pathological characteristic of type 2 diabetes and the elderly often develop insulin resistance. However, the deep-seated mechanisms for aging-related insulin resistance remain unclear. Here, we showed that nano-sized exosomes released by bone marrow mesenchymal stem cells (BM-MSCs) of aged mice could be taken up by adipocytes, myocytes, and hepatocytes, resulting in insulin resistance both in vivo and in vitro. Using microRNA (miRNA) array assays, we found that the amount of miR-29b-3p was dramatically increased in exosomes released by BM-MSCs of aged mice. Mechanistically, SIRT1 (sirtuin 1) was identified to function as the downstream target of exosomal miR-29b-3p in regulating insulin resistance. Notably, utilizing an aptamer-mediated nanocomplex delivery system, down-regulated the level of miR-29b-3p in BM-MSCs-derived exosomes significantly ameliorated the insulin resistance of aged mice. Meanwhile, BM-MSCs-specific overexpression of miR-29b-3p induced insulin resistance in young mice. Taken together, these findings suggested that BM-MSCs-derived exosomal miR-29b-3p could modulate aging-related insulin resistance, which may serve as a potential therapeutic target for aging-associated insulin resistance.

KEY WORDS: Insulin resistance, BM-MSCs, Exosome, microRNA, SIRT1

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Recently, the occurrence of type 2 diabetes increased steeply in elderly people, with about 25% of the elderly meeting the diagnostic criteria for type 2 diabetes.1 There is a clear association between type 2 diabetes and aging. However, the potential mechanisms have not been fully investigated yet.

Bone has many functions, such as supporting the body, protecting our internal organs from injury, and contributing to hematopoiesis.2, 3 Recently, bone has been revealed to function as an endocrine organ that regulates whole body metabolism systemically.4 Fibroblast growth factor 23 (FGF23), which is predominately secreted by osteocytes and/or osteoblasts,5 is a strong predictor of insulin resistance in people with chronic kidney disease.6 Osteocalcin, which is a protein synthesized by osteoblasts, exerts important roles in regulating whole body glucose and energy homeostasis.7-11 The osteoblast-derived lipocalin-2 (LCN2) has been revealed to regulate appetite by binding to melanocortin 4 receptor (MC4R) in the hypothalamus.12 In addition, BM-MSCs in bones have also been reported to function in liver injury,13 ganglion cells survival,14 and glial cell damage via diverse mechanisms.15 However, whether BM-MSCs also participate in the regulation of aging-associated insulin resistance remains unknown.

Exosomes are small extracellular vesicles released by multiple types of cells or tissues,16,17 and are important mediators of intercellular communications.18 Exosomes regulate the function of recipient cells or tissues by delivering effectors, such as proteins, mRNAs, or microRNAs (miRNA).19-21 MSCs generate higher amounts of exosomes than other cell types, such as myoblasts, acute monocytic leukemia cells, or embryonic kidney cells,22 and miRNAs in metabolic tissues play crucial roles in regulating glucose homeostasis.23 This prompted us to explore whether BM-MSCs are involved in regulating aging-associated insulin resistance via exosomal miRNAs or not.

Here, our findings revealed that the BM-MSCs-derived exosomes could be taken up 3


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by adipocytes, myocytes, and hepatocytes, respectively. Exosomes released by BM-MSCs of aged mice are able to cause insulin resistance both in vivo and in vitro. Mechanistically, we found the amount of miR-29b-3p was significantly increased in exosomes released by BM-MSCs of aged mice, and that Sirt1 (sirtuin 1) functions as the downstream target of exosomal miR-29b-3p in the regulation of insulin resistance. Notably, nanocomplex-mediated miR-29b-3p overexpression or inhibition in BM-MSCs-derived exosomes significantly impaired or enhanced mouse insulin sensitivity. Taken together, the present study revealed a role of BM-MSCs-derived exosomes in regulating aging-associated insulin resistance, and suggested that miR-29b-3p in exosomes released by BM-MSCs maybe a promising target for the therapy of aging-related insulin resistance.

RESULTS AND DISCUSSION The isolation and identification of BM-MSCs-derived exosomes To obtain BM-MSCs-derived exosomal particles, BM-MSCs were sorted out from bone marrow cells of 3-month old young mice and 18-month old aged mice using fluorescence activated cell sorting. The BM-MSCs were then cultured in exosome-free medium for 72 hours. Next, the culture medium was collected and the BM-MSCs-derived extracellular particles were isolated by ultracentrifugation. To identify whether the isolated extracellular particles were exosomes, first, their morphology was observed using transmission electron microscopy, which revealed that the particles were round-shaped vesicles with a double-layered membrane structure (Figure 1A). Second, the concentration and size distribution of the purified extracellular particles were measured using a nanoparticle tracking system, which demonstrated that the concentration of the particles was 2.6 × 109/mL, and the peak diameter was 115 nm (Figure 1B). Finally, western blotting results showed that the extracellular particles released by BM-MSCs of young mice and aged mice were positive for exosomal specific markers Syntenin 1 and tumor susceptibility gene 101 (TSG101), and extracellular vesicle (EV)-related protein CD63 (Figure 1C). Together, these results suggested that the BM-MSCs-derived extracellular particles collected in 4


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this study were exosomes.

Interestingly, the size and overall production of exosomes released by BM-MSCs of young mice and aged mice had no significant differences (Figure S1).

Exosomes released by BM-MSCs of aged mice impaired cellular insulin sensitivity in vitro Given that elderly people are prone to insulin resistance,24,25 we explored whether the exosomes released by BM-MSCs of aged mice participate in the regulation of insulin sensitivity. First, we tested whether BM-MSCs-derived exosomes could be taken up by adipocytes, myocytes, or hepatocytes. The BM-MSCs-derived exosomes were marked with red fluorescent dye PKH26, then co-cultured with 3T3-L1 adipocytes, C2C12 myocytes, or primary cultured hepatocytes. 12 hours later, these cells exhibited efficient uptake of BM-MSCs-derived exosomes, as evidenced by the existence of red fluorescence inside these cells (Figure 2A). Furthermore, we evaluated the effects of aged mice BM-MSCs-derived exosomes on cellular insulin sensitivity. Aged mice BM-MSCs-derived exosomes treatment significantly inhibited the insulin-stimulated glucose uptake of 3T3-L1 adipocytes and C2C12 myocytes (Figure 2B,C), while, the glucose output of primary cultured hepatocytes increased (Figure 2D). In addition, the insulin-stimulated activation of insulin receptor (IR), AKT, and glycogen synthase kinase 3β (GSK3β) phosphorylation in 3T3-L1 adipocytes, C2C12 myocytes, and primary cultured hepatocytes were all inhibited by aged mice BM-MSCs-derived exosomes treatment (Figure 2E-G). Taken together, these results suggested that aged mice BM-MSCs-derived exosomes impaired cellular insulin sensitivity of adipocytes, myocytes, and hepatocytes in vitro.

Administration of exosomes released by BM-MSCs from aged mice impaired insulin sensitivity in vivo To further explore the influences of BM-MSCs-derived exosomes on insulin sensitivity in vivo, insulin-sensitive young wild-type C57/BL6J mice (2 months old) 5


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were administered with the exosomes released by BM-MSCs from aged mice or young mice via tail vein injection (two times, 30 μg each time, 7 days apart). Consistent with the in vitro results, administration of aged mice BM-MSCs-derived exosomes significantly increased the fasting blood glucose levels, fasting serum insulin, and homoeostasis model assessment of insulin resistance (HOMA-IR) index of young wild-type C57/BL6J mice (Figure 3A-C). However, the fed blood glucose levels, fed serum insulin, body weight, and daily food intake had no obvious changes (Figure S2). Consistently, the glucose tolerance and clearance of young wild-type C57/BL6J mice were also significantly abrogated by the treatment of aged mice BM-MSCs-derived exosomes, as shown by the results of glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) (Figure 3D,E). Furthermore, the activation of insulin signaling in epididymal white adipose tissue (eWAT), muscle, and liver of young wild-type C57/BL6J mice induced by insulin stimulations was also inhibited by the treatment of aged mice BM-MSCs-derived exosomes, as evidenced by the immunostaining of phospho (p)-IR, p-AKT, and p-GSK3β in these tissues (Figure 3F-H).

Thus,

these

results

suggested

that

treatment

with

aged

mice

BM-MSCs-derived exosomes impaired insulin sensitivity in vivo.

The expression pattern of miRNAs in BM-MSCs-derived exosomes during aging Exosomes regulate a large number of physiological activities via exosomal miRNAs.26,27 Thus, we explored whether BM-MSCs-derived exosomes are involved in regulating aging-associated insulin resistance also through exosomal miRNAs or not. First, an miRNA microarray analysis was conducted to identify the differentially abundant miRNAs between BM-MSCs-derived exosomes from young mice and aged mice (Figure 4A). Among them, we focused on miR-29b-3p, the level of which was significantly increased in exosomes released by BM-MSCs of aged mice when compared with that from BM-MSCs of young mice (Figure 4B). Interestingly, treatment 3T3-L1 adipocytes, C2C12 myocytes, and primary cultured hepatocytes with aged mice BM-MSCs-derived exosomes significantly increased the level of miR-29b-3p in these cells (Figure 4C-E). Notably, in exosomes released by 6


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BM-MSCs of different aged humans, the abundance of miR-29b-3p correlated positively with increasing age (Figure 4F). Thus, these results suggested that the amount of miR-29b-3p increases in BM-MSCs-derived exosomes during aging.

Enhancing the expression of miR-29b-3p impaired cellular insulin sensitivity To investigate whether miR-29b-3p participates in regulating cellular insulin sensitivity, 3T3-L1 adipocytes, C2C12 myocytes, and primary cultured hepatocytes were transfected with miR-29b-3p mimics or miR-NC mimics (control). MiR-29b-3p overexpression significantly inhibited the insulin-stimulated glucose uptake of 3T3-L1 adipocytes and C2C12 myocytes (Figure 5A,B), while the glucose output of primary cultured hepatocytes was increased by miR-29b-3p mimics transfection (Figure 5C). Furthermore, we also measured the influences of miR-29b-3p overexpression on insulin-stimulated activation of cellular insulin signaling. In 3T3-L1 adipocytes, C2C12 myocytes, and primary cultured hepatocytes, the increased phosphorylation of IR, AKT, and GSK3β induced by insulin stimulations were all abrogated by miR-29b-3p mimics transfection (Figure 5D-F). Thus, these findings suggested that miR-29b-3p is a negative regulatory factor of cellular insulin sensitivity.

miR-29b-3p directly targets SIRT1 MiRNAs exert their functions by interacting with the 3' untranslated region (3' UTR) or protein coding sequence of target mRNAs.28 To search for the downstream effectors of miR-29b-3p in regulating insulin sensitivity, bioinformatic software, such as, miRanda, PicTar, and TargetScan were used to predict the possible targets of miR-29b-3p. Among the predicted target genes, SIRT1 was chosen as the candidate, because sequence analysis revealed there is a conserved miR-29b-3p binding site in the 3' UTR of SIRT1 mRNA (Figure 6A), and SIRT1 has been reported to play crucial roles in regulating insulin sensitivity.29 To explore the association between SIRT1 and miR-29b-3p, luciferase reporter plasmid containing the wild-type 3' UTR of SIRT1 (pGL3-SIRT1WT-3’UTR) was generated. In HEK293 cells co-transfected with 7


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pGL3-SIRT1WT-3’UTR reporter plasmids and miR-29b-3p mimics, miR-29b-3p overexpression significantly inhibited the luciferase activity (Figure 6B). However, this inhibition was largely abolished when two key nucleotides in the putative binding site of miR-29b-3p were mutated (Figure 6A,B). This result suggested that SIRT1 maybe the downstream effector of miR-29b-3p in the regulation of insulin sensitivity.

Furthermore, we measured the expression of SIRT1 in 3T3-L1 adipocytes, C2C12 myocytes, and primary cultured hepatocytes transfected with miR-29b-3p mimics or miR-NC mimics. MiR-29b-3p overexpression significantly decreased the protein levels of SIRT1 (Figure 6C). Consistent with the increased amount of miR-29b-3p in exosomes released by BM-MSCs of aged mice, the protein levels of SIRT1 in eWAT, muscle, and liver of aged mice (18 months) were also decreased when comparing to that in young mice (Figure 6D). Thus, these results further verified that SIRT1 is the downstream target of miR-29b-3p.

Moreover, we further explored whether miR-29b-3p regulates aging-associated insulin resistance via SIRT1 or not. In 3T3-L1 adipocytes, C2C12 myocytes, and primary cultured hepatocytes, the decreased insulin sensitivity induced by the transfection of miR-29b-3p mimics could be rescued by SIRT1 overexpression (Figure

6E-G).

Interestingly,

miR-29b-3p

overexpression-induced

decreased

insulin-stimulated glucose uptake of 3T3-L1 adipocytes and C2C12 myocytes, and increased glucose output of primary cultured hepatocytes were all rescued by the treatment of resveratrol (100 μM), which is a classical agonist of SIRT1 (Figure 6H-J). Thus, all these results suggested that SIRT1 is the direct downstream effector of miR-29b-3p in the regulation of insulin sensitivity.

Nanocomplex mediated BM-MSCs-specific overexpression of miR-29b-3p impaired the insulin sensitivity To explore the role of miR-29b-3p in BM-MSCs-derived exosomes in regulating insulin sensitivity, a BM-MSCs-specific nanocomplex/aptamer-agomiR-29b-3p was 8


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injected to the bone marrow cavity of young mice.30,31 qRT-PCR analysis confirmed that intra-bone marrow injection of aptamer-agomiR-29b-3p largely increased the amount of miR-29b-3p in BM-MSCs-derived exosomes (Figure 7A). Furthermore, we studied the effects of miR-29b-3p overexpression in BM-MSCs-derived exosomes on insulin

sensitivity.

In

mice

injected

with

BM-MSCs-specific

nanocomplex/aptamer-agomiR-29b-3p, the fasting blood glucose levels, fasting serum insulin, and HOMA-IR index were all significantly increased (Figure 7B-D), while the fed blood glucose levels, fed serum insulin, body weight, and daily food intake did not changed when compared with those in the control mice (Figure S3). The GTTs and ITTs results demonstrated that the glucose tolerance and clearance of mice were all decreased after administration of BM-MSCs-specific aptamer-agomiR-29b-3p (Figure 7E,F). In addition, the insulin-stimulated activation of insulin signaling in eWAT, muscle, and liver of the mice was also significantly inhibited by BM-MSCs-specific aptamer-agomiR-29b-3p injection (Figure 7G-I). Together, all these findings suggest that overexpression of miR-29b-3p in BM-MSCs-derived exosomes impaired the insulin sensitivity of mice in vivo.

Nanocomplex-mediated BM-MSCs-specific inhibition of miR-29b-3p alleviates aging-associated insulin resistance The findings above suggested that miR-29b-3p in exosomes released by BM-MSCs from aged mice could be a potential therapeutic target for aging-associated insulin resistance. To investigate the therapeutic potential of targeting miR-29b-3p in BM-MSCs-derived

exosomes

for

aging-associated

insulin

resistance,

BM-MSCs-specific nanocomplex/aptamer-antagomiR-29b-3p was injected to the femoral bone marrow cavity of 15 months old wild-type C57/BL6J mice (twice a month for three months). Injection of aptamer-antagomiR-29b-3p significantly decreased the amount of miR-29b-3p in BM-MSCs-derived exosomes (Figure 8A). Notably, the level of miR-29b-3p in serum-derived exosomes was also decreased (Figure 8B). In previous studies, exosomes released by human umbilical cord MSCs (HucMSC-Exos) were administered to animals via intravenous injection.32 9


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Intravenous injection is a relatively non-invasive method compared with intra-bone marrow injection; however, the delivery efficiency of aptamer-antagomiR-29b-3p to BM-MSCs of mice was higher when delivered by intra-bone marrow injection compared with that achieved by intravenous injection (Figure S4). Furthermore, we evaluated the effects of BM-MSCs-specific aptamer-antagomiR-29b-3p injection on the aging-associated insulin resistance. Mice intra-bone marrow injected with BM-MSCs-specific aptamer-antagomiR-29b-3p exhibited lower fasting blood glucose levels, lower fasting serum insulin, and a decreased HOMA-IR index when compared with those in the control mice (Figure 8C-E). Meanwhile, the fed blood glucose levels, fed serum insulin, body weight, and daily food intake did not changed (Figure S5). Moreover, the glucose tolerance and clearance of aged mice were significantly enhanced by BM-MSCs-specific aptamer-antagomiR-29b-3p injection, as shown by the results of GTTs and ITTs (Figure 8F,G). Consistently, the insulin-stimulated activation of insulin signaling in eWAT, muscle, and liver of the aged mice was also augmented

by

BM-MSCs-specific

aptamer-antagomiR-29b-3p

injection,

as

demonstrated by the immunostaining of p-IR, p-AKT, and p-GSK3β in these tissues (Figure 8H-J). Taken together, all these results suggested that nanocomplex-mediated targeting miR-29b-3p

in

BM-MSCs-derived

exosomes could

alleviate the

aging-associated insulin resistance.

In the literature, it has been reported that HucMSC-Exos could restore the degenerated function of pancreas in diabetes mellitus animal models.32 However, in this

study,

the

results

showed

that

BM-MSCs-specific

nanocomplex/aptamer-antagomiR-29b-3p-mediated inhibition the accumulation of miR-29b-3p in BM-MSCs-derived exosomes had no obvious impacts on aging-associated degeneration of pancreatic function (Figure S6). This suggested that the exosomes released by BM-MSCs are different from HucMSC-Exos, it may not exert crucial effects on aging-related degeneration of pancreatic function.

10


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Insulin resistance is a complex metabolic disorder to which the elderly are prone. However, the underlying mechanisms are unclear. Previous studies mainly focused on major metabolic tissues, such as, liver, muscle, and adipose tissue, in the regulation of glucose

metabolism

and

insulin

sensitivity.33-36

Here,

we

showed

that

BM-MSCs-derived exosomes also participate in regulating aging-associated insulin resistance.

In this study, we found BM-MSCs could communicate with the liver, muscle, and adipose tissue via secreting and delivering of exosomes. Thus, the altered information in BM-MSCs of aged mice could be transmitted to the liver, muscle, and adipose tissue. The BM-MSCs-derived exosomes play crucial roles in mediating the regulation of aging-associated insulin resistance.

Exosomes are small extracellular vesicles released by multiple types of cells or tissues,16,17 they are important mediators of intercellular communications. Recent findings have revealed that BM-MSCs can communicate with other cells or tissues by secreting exosomes. For example, Chen et al. reported that exosomes released by BM-MSCs could protect against liver injury,13 and BM-MSCs-derived exosomes could enhance the survival of retinal ganglion cells.14 In this study, we showed that aged mice BM-MSCs-derived exosomes are able to cause insulin resistance in vivo. In addition, the insulin-stimulated activation of insulin signaling in 3T3-L1 adipocytes, C2C12 myocytes, and primary cultured hepatocytes were also significantly inhibited by aged mice BM-MSCs-derived exosomes treatment. All these results suggest that aged mice BM-MSCs-derived exosomes impair insulin sensitivity both in vivo and in vitro.

MiRNAs are small, noncoding regulatory RNA molecules.28 In this study, we revealed that the abundance of miR-29b-3p is largely increased in exosomes released by BM-MSCs of aged mice, and the BM-MSCs-derived exosomes could be taken up by 3T3-L1 adipocytes, C2C12 myocytes, and primary cultured hepatocytes. In 11


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adipocytes, myocytes, and hepatocytes, miR-29b-3p binds to the 3' UTR of SIRT1 mRNA and inhibits its expression directly. SIRT1 is a well-known insulin sensitive gene.37,38 The aged mice BM-MSCs-derived exosomes regulate aging-associated insulin resistance in a miR-29b-3p/SIRT1-dependent manner. In fact, the association between miR-29b-3p and SIRT1 has already been reported previously.39, 40 But, a role of miR-29b-3p/SIRT1 signaling in the regulation of insulin resistance, especially aging-related insulin resistance, was unknown. Our study discovered that exosomes released by BM-MSCs of aged mice could participate in the regulation of aging-associated insulin resistance via the miR-29b-3p/SIRT1 signaling pathway.

Interestingly, in exosomes released by BM-MSCs from humans at different ages, the levels

of

miR-29b-3p

correlated

positively

with

age

increase.

Notably,

aptamer-antagomiR-29b-3p-mediated inhibition of miR-29b-3p in BM-MSCs-derived exosomes significantly ameliorated the insulin resistance of aged mice. Meanwhile, aptamer-agomiR-29b-3p-mediated increased miR-29b-3p in BM-MSCs derived exosomes had the opposite effect. In previous studies, Sun Y et al. identified that HucMSC-Exos could restore the degenerated function of pancreas in a rat model of diabetes mellitus.32 However, in this study, our findings showed that inhibition of miR-29b-3p in BM-MSCs-derived exosomes had no significant effects on age-related degeneration of pancreatic function. Thus, BM-MSCs-derived exosomal miR-29b-3p plays crucial roles in regulating aging-associated insulin resistance without affecting the degenerated function of the pancreas.

CONCLUSION In conclusion, the present study identified a role of BM-MSCs-derived exosomes in regulating aging-associated insulin resistance. MiR-29b-3p in BM-MSCs-derived exosomes could represent as a promising target for the therapy of aging-associated insulin resistance.

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METHODS Animals and treatment: all animals care and experimental protocols were reviewed and approved by the Animal Care and Use Committees of the Laboratory Animal Research Center at Xiangya Medical School of Central South University. All animals used in this study were maintained in specific pathogen-free facility of the Laboratory Animal Research Center at Central South University, with free access to food and water prior to the initiation of experiments.

Cell culture and treatment: mouse BM-MSCs were isolated and cultured as discribed previously.41 Briefly, bone marrow cells were ﬂushed out from the bone marrow cavity of 3-month old young mice and 18-month old aged mice, then, incubated at 4°C for twenty minutes with the following antibodies: Sca-1, CD29, CD45,

and

CD11b,

all

purchased

from

BioLegend.

Next,

the

Sca-1+CD29+CD45–CD11b– BM-MSCs were sorted out by flow cytometry (BD Biosciences) and cultured at 37°C with the supplement of 5% CO2. Culture medium composed of DMEM, streptomycin/penicillin and 10% exosome-free FBS was used for the culture of BM-MSCs in this study.

Primary hepatocytes were isolated and cultured as reported previously.42,43 Briefly, mice were anesthetized and pre-perfused with a calcium free HEPES phosphate buffer A for 5 min. Then, collagenase-containing buffer B was continually infused. Until the appearance of corrugation on the surface of the liver, the perfusion was stopped and the cells in the digested liver were teased out, filtered and centrifuged. After the removal of death cells by Percoll (sigma), the hepatocytes were counted and seeded in 12-well plates with DMEM (Gibco) containing 10 nM dexamethasone, 10% FBS and antibiotics.

C2C12 myoblasts and 3T3-L1 pre-adipocytes were cultured in a humidified incubator at the temperature of 37°C and supplemented with 5% CO2. Standard protocols were utilized to induce the differentiation of these cells.44 13


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For the transfection of miR-29b-3p mimics or miR-NC mimics, 3T3-L1 adipocytes, C2C12 myocytes and primary cultured hepatocytes were seeded in 12-well plates and transfected with lipofectamine 2000 (Thermo Scientific). For the overexpression of SIRT1, primary cultured hepatocytes, 3T3-L1 adipocytes and C2C12 myocytes were infected with SIRT1 overexpressing adenoviruses or lentiviruses, respectively.

Exosomes isolation, identification and treatment: after culture in exosome-free medium for 72 hours, the culture medium of BM-MSCs was collected and centrifuged to remove the death cells or cell debris. Next, the supernatant was filtrated with 0.22 μm filter and subjected to ultracentrifugation at 100,000 g for 4-6 hours at 4°C. The exosomes were congregated at the bottom of tube.

To identify the isolated exosomes, western blotting analysis was conducted to detect the expression of TSG101, Syntenin 1 and CD63. NanoSight analysis (Particle Metrix, Meerbusch, Germany) was performed to measure the size distribution and concentration of isolated particles released by BM-MSCs. To observe the morphology of BM-MSCs derived particles, transmission electron microscopy imaging was carried out (Hitachi H7500 TEM, Tokyo,Japan).

For the administration of exosomes to mice, BM-MSCs derived exosomes were injected to recipient mice via tail vein injection (for two times, 30 μg/time in every 7 days). For the cell treatment, exosomes were added to the culture medium on the basis of 2 μg of exosomes per 1×105 recipient cells. To monitor the exosome trafficking, exosomes were marked by a PKH26 fluorescent cell linker kit (Sigma), according to the manufactor's instructions.

Immuno-blotting and qRT-PCR analysis: western blotting analysis was conducted as previously described.45 Primary antibodies: p-IR (CST3024s), IR (CST3025s), p-AKT (CST9271s), AKT (CST9272s), p-GSK3β (CST9336s) and GSK3β (CST9315s) were all purchased from Cell Signaling Technology, CD63 (ab68418) 14


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and TSG101 (ab125011) were purchased from Abcam, and syntenin 1 (PA5-28826) was purchased from ThermoFisher Scientific. All the primary antibodies were incubated at 4 °C overnight and specific proteins were visualized by ECL Plus. For qRT-PCR analysis, the total RNAs from cultured cells were isolated by Trizol (Thermo Fisher Scientific). The exosomal miRNAs were extracted using a Qiagen miRNeasy Mini Kit.

Blood glucose, serum insulin, GTTs, ITTs and HOMA-IR index: the measurement of blood glucose levels, serum insulin, and the operation of GTTs and ITTs were performed as reported previously.46,47 The following formula was used to calculate the HOMA-IR index: [fasting blood glucose levels (mmol/L)] x [fasting serum insulin levels (μU/ml)]/ 22.5.

The glucose uptake and glucose output assay: the glucose uptake and glucose output assays were performed as described previously.44 The glucose levels in medium were measured by a glucose assay kit (Abcam, ab65333), according to the manufacture's instructions.

miRNA microarray assay: to identify the differently expressed miRNAs in the exosomes released by BM-MSCs from aged mice and young mice. miRNA microarray assays were performed under the help of Obio Technology (Shanghai) Corp.,Ltd.

Luciferase activity assay: luciferase activity assays were conducted as reported previously.30 The wild-type SIRT1 3' UTR firefly luciferase reporter plasmids and SIRT1 3' UTR firefly luciferase reporter plasmids with the potential miR-29b-3p binding site mutated were co-transfected with miR-29b-3p mimics or miR-NC mimics to HEK293 cells, respectively. Renilla luciferase reporter plasmids were also transfected as internal control. 48 hours post transfections, the luciferase activities were measured by a Dual-Glo Luciferase Assay Kit (Promega). 15


Page 16 of 38

Page 17 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Construction of BM-MSCs specific aptamer delivery system: the construction of BM-MSCs specific aptamer mediated nanocomplex delivery system was performed as described previously.30 The aptamer sequence that had highest affinity to BM-MSCs was 5’-GAATTCAGTCGGACAGCGACGACGGTGATATGTCA AGGTCGTATGCACGAGTCAGAGGGATGGACGAATATCGTCTCCC-3’.

The

BM-MSCs

and

specific

nanocomplex/aptamer-agomiR-29b-3p

nanocomplex/aptamer-antagomiR-29b-3p were generated as following: Six parts of 4.2 μM sodium citrate was mixed with one part of polyethyleneimine (PEI) solution (100 μg/ml, pH=6.0) to generate PEI-citrate nanocore. Then, three parts of aptamer (50 nM) and agomiR-29b-3p (1 μM) or antagomiR-29b-3p (1 μM) were added into the nanocore to assemble nanocomplex. nanocomplex/aptamer-agomiR-29b-3p or nanocomplex/aptamer-antagomiR-29b-3p, or a comparable volume of control (20 μl) was injected to the medullary cavity of mice via intra-bone marrow injection, twice a month for 3 months. The collection of human bone marrow samples: before bone marrow aspiration and collection, all participants were received written informed consent. There were 52 patients, including 30 males and 22 females, who underwent hip replacement were participated in this study. The age of the participants was range from 20 to 79 years old. The clinical studies were approved by the Ethics Committee of Xiangya Hospital at Central South University. Quantification and statistical analysis: all data are presented as means ± S.E.M. The statistical significance between various groups or treatments was measured by Student’s t test or ANOVA followed by Bonferroni post-test. Data analyses were carried out by GraphPad Prism 7.0. p < 0.05 was considered statistically significant.

16


Figures

A

Page 18 of 38

Figure 1 B

C 5.0

Particle count (x106)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

4.0

3 mo

3.0

Syntenin 1

2.0

TSG101

1.0 0.0

CD63 0

100 200 300 400 500

Particle size (nm)

17 ACS Paragon Plus Environment

18 mo

Page 19 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 1. Isolation and identification of BM-MSCs derived exosomes (A) Representative picture of the ultrastructure of BM-MSCs derived exosomes, scale bar: 100 nm; (B) The size distribution profile of BM-MSCs derived exosomes; (C) The protein levels of TSG101, syntenin 1 and CD63 in exosomes released by BM-MSCs from 3 months old young mice and 18 months old aged mice.

18


A PKH26-labeled Exos / Nucleus

C

D

3T3-L1 adipocytes

*

100 50 0

-

BM-MSC-Exos BM-MSC-Exos insulin

E

-

+ - - + - - +

+ +

100 50 0

+ - - + - + - - + - - + + +

*

F

-

+ - - + - + - - + - - + + +

-

+ - - + - - +

+ +

100 50

Basal

G + +

t-IR

t-IR

t-IR

p-AKT

p-AKT

p-AKT

t-AKT

t-AKT

t-AKT

p-GSK3β

p-GSK3β

p-GSK3β

t-GSK3β

t-GSK3β

t-GSK3β

*

*

1.0

0.5

0.0

* 1.5

p-IR

p-AKT

p-GSK3β

*

*

*

*

0.5

0.0

*

*

1.0

ACS Paragon Plus Environment 19

p-IR

p-AKT

p-GSK3β

GCG/Ins

+ +

+ +

Empty liposome + ins Young BM-MSCs-Exos + ins Aged BM-MSCs-Exos + ins

1.5 Arbitrary Units

*


Arbitrary Units

1.5

*

+ - - + - - +

p-IR

*

GCG

Primary hepatocytes

p-IR

*

*

150

p-IR


*

200

0

C2C12 myocytes

+ +

*

250

150

Young BM-MSC-Exos Aged BM-MSC-Exos insulin

3T3-L1 adipocytes BM-MSC-Exos BM-MSC-Exos insulin

*

200

Empty liposome Young BM-MSCs-Exos Aged BM-MSCs-Exos

Glucose output (% )

150

Primary hepatocytes

C2C12 myocytes

Glucose uptake (% )

*

200

Arbitrary Units

primary hepatocytes

C2C12 myocytes

3T3-L1 adipocytes

B

Glucose uptake (% )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Young 38 39 Aged 40 41 42 43 44 45 46 Young 47 48 Aged 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 38 Figure 2

ACS Nano

*

*

*

*

*

1.0

0.5

0.0

p-IR

p-AKT

p-GSK3β

Page 21 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 2. Aged mice BM-MSCs derived exosomes impaired cellular insulin sensitivity in vitro (A) BM-MSCs derived exosomes were marked with red flurescence dye PKH26 and then co-cultured with 3T3-L1 adipocytes, C2C12 myocytes, and primary cultured hepatocytes, red flurescence represents exosomes in these cells as indicated by white arrows, scale bar: 25μm; (B-D) Effects of BM-MSCs derived exosomes on glucose uptake of 3T3-L1 adipocytes and C2C12 myocytes as well as glucose output of primary cultured hepatocytes; (E-G) The insulin stimulated IR, AKT, and GSK3β phosphorylation in 3T3-L1 adipocytes, C2C12 myocytes, and primary cultured hepatocytes treated with BM-MSCs derived exosomes as indicated (top: representative pictures of western blot, bottom, quantitative measurements of p-IR, p-AKT and p-GSK3β proteins ). All these experiments were repeated for three times and the data are presented as means ± SEM. Statistical significance was calculated by two-tailed Student's t test (*P < 0.05).

20


B

150 100 50 0

D 500

*

300 200 100 0

*

* 0

30

F

*

0.0

E

+ - - + - - +

*

150 100

*

0

30

+ +

60 90 Time (min)

-

+ +

+ - - + - - +

+ +

p-IR

t-IR

t-IR

t-IR

p-AKT

p-AKT

p-AKT

t-AKT

t-AKT

t-AKT

p-GSK3β

p-GSK3β

p-GSK3β

t-GSK3β

t-GSK3β

t-GSK3β

*

*

Control + ins Young BM-MSCs-Exos + ins Aged BM-MSCs-Exos + ins

*

1.5

*

*

*

*

*

1.0

0.5

0.5

p-IR

p-AKT

p-GSK3β

0.0


p-IR

p-AKT

p-GSK3β

+ +

+ +


1.5

Arbitrary Units

*

*

+ - - + - - +

-

+ +

p-IR

*

120

Liver

p-IR

*

*

*

*

H

Arbitrary Units

Arbitrary Units

1

Muscle

1.0

0.0

2

50 0

120


1.5

3

Control Young BM-MSC-Exos Aged BM-MSC-Exos

200

eWAT

-

*

0

G BM-MSC-Exos BM-MSC-Exos insulin

*

4

0.2

*

60 90 Time (min)

C

0.4

Control Young BM-MSC-Exos Aged BM-MSC-Exos

*

400

Page 22 of Figure 338

HOMA-IR index

*

*

0.6

Blood glucose level (mg/dL)

*

200

Fasting serum insulin (ng/ml)

Fasting blood glucose level (mg/dL)

A


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Young 48 Aged 49 50 51 52 53 54 55 56 57 58 59 60

Control ACS Nano Young BM-MSCs-Exos Aged BM-MSCs-Exos

*

*

*

*

*

*

1.0

0.5

0.0

p-IR

p-AKT

p-GSK3β

Page 23 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 3. Aged mice BM-MSCs derived exosomes impaired the insulin sensitivity in vivo (A-C) The fasting blood glucose levels, fasting serum insulin, and HOMA-IR index of young wild-type C57/BL6J mice (2 months old) administrated with Aged BM-MSC-Exos or Young BM-MSC-Exos or Control as indicated (for two times, 30 μg/time in every 7 days); (D-E) The GTTs and ITTs in young wild-type C57/BL6J mice injected with Aged BM-MSC-Exos or Young BM-MSC-Exos or Control; (F-H) The insulin stimulated IR, AKT, and GSK3β phosphorylation in epididymal white adipose tissue (eWAT), muscle and liver of mice injected with Aged BM-MSC-Exos or Young BM-MSC-Exos or Control as indicated (top: representative pictures of western blot, bottom, quantitative measurements of p-IR, p-AKT and p-GSK3β proteins ). Data are presented as means ± SEM (n= 6-7). Statistical significance was calculated by two-tailed Student's t test or two-way ANOVA, (*P < 0.05).

22



ACS Nano

C Young BM-MSC-Exos Aged BM-MSC-Exos

*

+ Empty liposome + Young BM-MSCs-Exos + Aged BM-MSCs-Exos

*

6 miR-29b-3p level (fold)

miR-29b-3p level (fold)

6

D

4

2

0

*

*

5

4

2

0

+ Empty liposome + Young BM-MSCs-Exos + Aged BM-MSCs-Exos

miR-29b-3p level (fold)

B

3T3-L1 adipocytes

*

4 3 2 1 0

C2C12 myocytes

F

E + Empty liposome + Young BM-MSCs-Exos + Aged BM-MSCs-Exos

*

4 3 2 1 0 Primary hepatocytes



*


A

1 2 3 4 5 Young BM-MSC-Exos Aged BM-MSC -Exos 6 7 8 mmu−miR−17−5p 9 mmu−miR−762 10 11 mmu−miR−465b−5p 12 mmu−miR−221−3p 13 14 mmu−miR−6409 15 mmu−miR−151−5p 16 17 mmu−miR−29b−3p 18 mmu−miR−191−5p 19 20 mmu−miR−99b−5p 21 mmu−miR−7118−5p 22 23 mmu−miR−6347 24 mmu−miR−290b−3p 25 mmu−let−7a−5p 26 27 mmu−miR−3107−5p 28 mmu−miR−486−5p 29 30 mmu−miR−107−3p 31 mmu−miR−24−3p 32 33 mmu−miR−1931 34 mmu−miR−92b−3p 35 36 mmu−miR−20a−5p 37 mmu−miR−6968−5p 38 mmu−miR−7025−5p 39 40 mmu−miR−6538 41 mmu−miR−190a−3p 42 43 mmu−miR−702−5p 44 45 46-1.5 -0.75 0 0.75 1.5 47 48 49 50 51 52 53 54 55 56 57 58 59 60

8 6 4 2 0 20

40

60 80 Age (year)

100

Page 25 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 4. The expression pattern of miRNAs in BM-MSCs derived exosomes during aging (A) miRNA microarray analyzes the differently expressed miRNAs in Aged BM-MSC-Exos and Young BM-MSC-Exos; (B) The amount of miR-29b-3p in Aged BM-MSC-Exos and Young BM-MSC-Exos, (C-E) The levels of miR-29b-3p in 3T3-L1 adipocytes, C2C12 myocytes, and primary cultured hepatocytes treated with Aged BM-MSC-Exos or Young BM-MSC-Exos or empty liposome as indicated. (F) The levels of miR-29b-3p in exosomes released by BM-MSCs of humans at different ages. Data are shown as means ± SEM (The cell experiments were repeated for three times). Statistical significance was calculated by two-tailed Student's t test, (*P < 0.05).

24


Page 26 of538 Figure

ACS Nano

A

B

C C2C12 myocytes

Glucose uptake (% )

Glucose uptake (% )

*

*

150 100 50 0

-

insulin miR-29b-3p mimics

+

+ -

200

100

Basal GCG GCG/Ins

F Primary hepatocytes

C2C12 myocytes miR-NC mimics miR-29b-3p mimics insulin

+ - + - -

+ +

+ +

p-IR

t-IR

t-IR

p-AKT

p-AKT

t-AKT

t-AKT

p-GSK3β

p-GSK3β

t-GSK3β

t-GSK3β

miR-NC mimics + ins miR-29b-3p mimics + ins 1.5

*

*

*

0.5

0.5

p-AKT p-GSK3β

0.0

p-IR

p-AKT


+ -

miR-NC mimics miR-29b-3p mimics insulin

p-IR

ins

*

* *

*

0

1.0

p-IR

300

+ +

E

1.0

0.0

200

Arbitrary Units

Arbitrary Units

1.5

*

250

D

- miR-29b-3p mimics + miR-29b-3p mimics Glucose output (% )

*

Primary hepatocytes

p-GSK3β

+ -

+ +

+ +

miR-NC mimics + ins miR-29b-3p mimics + ins 1.5

Arbitrary Units

1 2 3 3T3-L1 adipocytes 4 5 6 7 8 9 200 10 11 12 150 13 14 100 15 16 17 50 18 19 20 0 21 22 - - + + insulin 23 - + - + miR-29b-3p mimics 24 25 26 27 28 29 30 31 3T3-L1 adipocytes 32 33 34 miR-NC mimics + - + 35 - + + miR-29b-3p mimics 36 insulin + + 37 38 p-IR 39 40 41 t-IR 42 43 44 p-AKT 45 46 47 t-AKT 48 49 50 p-GSK3β 51 52 53 t-GSK3β 54 55 56 miR-NC mimics + ins 57 58 miR-29b-3p mimics + 59 60

*

*

*

1.0 0.5 0.0

p-IR

p-AKT p-GSK3β

Page 27 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 5. Enhancing the expression of miR-29b-3p impaired cellular insulin sensitivity (A-C) The impacts of miR-29b-3p overexpression on glucose uptake of 3T3-L1 adipocytes and C2C12 myocytes as well as glucose output of primary cultured hepatocytes; (D-F) The insulin stimulated IR, AKT, and GSK3β phosphorylation in 3T3-L1 adipocytes, C2C12 myocytes, and primary cultured hepatocytes transfected with miR-29b-3p mimics or miR-NC mimics as indicated (top: representative pictures of western blot, bottom, quantitative measurements of p-IR, p-AKT, and p-GSK3β proteins ). All these experiments were repeated for three times and the data are presented as means ± SEM. Statistical significance was calculated by two-tailed Student's t test (*P < 0.05).

26


A SIRT1 WT 3’UTR

533 556 5’- UGGAGCACUCAAAACUUGGUGCUC-3’

Mmu-miR-29b-3p

3’-UUGUGACUAAAGUUUACCACGAU-5’

SIRT1 mutant 3’UTR

5’- UGGAGCACUCAAAACUUGCUGCUC-3’

B

C SIRT1 WT 3’UTR + miR-NC mimics SIRT1 WT 3’UTR + miR-29b-3p mimics SIRT1 mutant 3’UTR + miR-29b-3p mimics

*

*

100

50

SIRT1

3T3-L1 adipocytes

GAPDH

GAPDH

SIRT1

SIRT1

C2C12 myocytes

GAPDH

GAPDH

SIRT1

SIRT1 Liver GAPDH

F

G

3T3-L1 adipocytes SIRT1 miR-29b-3p mimics Insulin

+

+ +

+ +

+ + +

Primary hepatocytes

C2C12 myocytes

+

SIRT1 miR-29b-3p mimics Insulin

+ +

+ +

+ + +

SIRT1 miR-29b-3p mimics Insulin

p-IR

p-IR

p-IR

t-IR

t-IR

t-IR

p-AKT

p-AKT

p-AKT

t-AKT

t-AKT

t-AKT

p-GSK3β

p-GSK3β

p-GSK3β

t-GSK3β

t-GSK3β

t-GSK3β

SIRT1

SIRT1

SIRT1

GAPDH

GAPDH

GAPDH

0

p-IR

H

p-AKT

p-GSK3β

3T3-L1 adipocytes

*

Glucose uptake (% )

250

*

200 150

*

*

100 50 0

insulin miR-29b-3p mimics Resveratrol

-

+ -

Arbitrary Units

* * * * * * *

* *

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3 2 1 0

*

*

* * * * * * * p-IR

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p-GSK3β

J

C2C12 myocytes

250

Glucose uptake (% )

Arbitrary Units

eWAT

Muscle

GAPDH

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1

o 18 m

Primary hepatocytes

0

2

nths

nths

3 mo

SIRT1

Arbitrary Units

Relative luciferase activity (%)

150

D

s imic ics m m i p 3 NC m iR-29bmiRm

200 150

*

*

*

*

250

100 50 0

- + + - + ACS Paragon - Environment - Plus - + + miR-29b-3p mimics Resveratrol 27- - + insulin

3 2 1 0

+

+ +

+ +

+ + +

* * * * * * * * * p-IR

p-AKT

p-GSK3β

Primary hepatocytes

- miR-29b-3p - Resveratrol + miR-29b-3p - Resveratrol + miR-29b-3p + Resveratrol

+ + +

Glucose output (% )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ACS Nano

200

*

*

* *

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Basal

GCG

GCG/Ins

Page 29 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 6. miR-29b-3p regulates cellular insulin sensitivity via SIRT1 (A) Schematic of the sequence that miR-29b-3p targets the WT or mutated 3' UTR of SIRT1 mRNA; (B) The luciferase activity of SIRT1 WT or mutated 3' UTR reporter plasmids in HEK293 cells co-transfected with miR-29b-3p mimics or miR-NC mimics as indicated; (C) The protein levels of SIRT1 in 3T3-L1 adipocytes, C2C12 myocytes, and primary cultured hepatocytes transfected with miR-29b-3p mimics or miR-NC mimics; (D) The expression of SIRT1 in eWAT, muscle, and liver of 3-month old young mice and 18-month old aged mice; (E-G) The insulin-stimulated IR, AKT, and GSK3β phosphorylation in 3T3-L1 adipocytes, C2C12 myocytes, and primary cultured hepatocytes infected with SIRT1 overexpression lentiviruses or adenoviruses and co-transfected with miR-29b-3p mimics or miR-NC mimics as indicated (top: representative pictures of western blot, bottom, quantitative measurements of p-IR, p-AKT and p-GSK3β proteins); (H-J) The effects of resveratrol (100 μM) on insulin-stimulated glucose uptake of 3T3-L1 adipocytes and C2C12 myocytes as well as glucose output of primary cultured hepatocytes transfected with miR-29b-3p mimics or miR-NC mimics as indicated. Data are shown as means ± SEM (n=6-7 in D and the cell experiments were repeated for three times). Statistical significance was calculated by two-tailed Student's t test or two-way ANOVA, (*P < 0.05).

28


Vehicle ACS Nano Aptamer-agomiR-NC Aptamer-agomiR-29b-3p

B

Relative miR-29b-3p level (fold)


*

C

*

150

100

50

0

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*

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*

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*

*

*

30

60 90 Time (min)

G



D Fasting serum insulin (ng/ml)

A

HOMA-IR index

1 2 3 4 5 6 7 10 8 9 10 11 12 13 5 14 15 16 17 18 19 0 20 21 22 23 24 25 26 27 500 28 29 30 400 31 32 300 33 34 35 200 36 37 100 38 39 0 40 0 41 42 43 44 45 46 47 48 49 50 Aptamer-agomiR-NC 51 52 Aptamer-agomiR-29b-3p 53 54 Insulin 55 56 p-IR 57 58 59 t-IR 60

Page 30 of 38

Figure 7

120

Vehicle Aptamer-agomiR-NC Aptamer-agomiR-29b-3p

250 200

*

150

*

100

*

*

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90

50 0

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Time (min)

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Aptamer-agomiR-NC Aptamer-agomiR-29b-3p Insulin

Aptamer-agomiR-29b-3p Insulin

t-IR

t-IR

p-AKT

p-AKT

p-AKT

t-AKT

t-AKT

t-AKT

p-GSK3β

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p-AKT p-GSK3β

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p-AKT ACS Paragon p-IR Plus Environment 29

p-GSK3β

Aptamer-agomiR-NC + ins Aptamer-agomiR-29b-3p + ins

Arbitrary Units

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Arbitrary Units

Arbitrary Units


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Aptamer-agomiR-NC

p-IR

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I

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p-IR

p-AKT p-GSK3β

Page 31 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 7. Nanocomplex/Aptamer mediated BM-MSCs specific overexpression of miR-29b-3p impaired the insulin sensitivity (A) The amounts of miR-29b-3p in exosomes released by BM-MSCs of ten-weeks old young

mice

intra–bone

marrow

nanocomplex/aptamer-agomiR-29b-3p

injected or

with

BM-MSCs

specific

nanocomplex/aptamer-agomiR-NC

or

vehicle for three months (twice a month); (B-D) The fasting blood glucose levels, fasting

serum

insulin,

and

HOMA-IR

index

of

mice

injected

with

aptamer-agomiR-29b-3p or aptamer-agomiR-NC or vehicle; (E-F) The GTTs and ITTs in mice injected with aptamer-agomiR-29b-3p or aptamer-agomiR-NC or vehicle; (G-I) Insulin-stimulated IR, AKT, and GSK3β phosphorylation in eWAT, muscle,

and

liver

of

mice

injected

with

aptamer-agomiR-29b-3p

or

aptamer-agomiR-NC, respectively (top: representative pictures of western blot, bottom, quantitative measurements of p-IR, p-AKT, and p-GSK3β proteins ). Data are presented as means ± SEM (n= 6-7). Statistical significance was calculated by two-tailed Student's t test or two-way ANOVA, (*P < 0.05).

30


B


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0* 0

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C 1.5

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Figure 8

Fasting serum insulin (ng/ml)

A


1 2 3 4 5 6 7 8 1.5 9 10 11 1.0 12 13 14 15 0.5 16 17 18 19 0.0 20 21 22 23 24 25 26 27 28 29 3 30 31 32 33 2 34 35 36 37 1 38 39 40 41 0 42 43 44 45 46 47 48 49 50 51 52 Aptamer-antagomiR-NC 53 Aptamer-antagomiR-29b-3p 54 55 Insulin 56 57 58 p-IR 59 60

Vehicle ACS Nano Aptamer-antagomiR-NC Aptamer-antagomiR-29b-3p

200 150 100

*

50 0

0

I

60

90

Muscle

Aptamer-antagomiR-NC Aptamer-antagomiR-29b-3p Insulin

Liver

+ - + - + - + - - + +

Aptamer-antagomiR-NC Aptamer-antagomiR-29b-3p Insulin

p-IR

p-IR

t-IR

t-IR

t-IR

p-AKT

p-AKT

p-AKT

t-AKT

t-AKT

t-AKT

p-GSK3β

p-GSK3β

p-GSK3β

t-GSK3β

t-GSK3β

t-GSK3β

*

*

1 0

3

*

2

*

*

1


p-IR

p-AKT p-GSK3β

31

0

p-IR

p-AKT

p-GSK3β

+ - + - + - + - - + +

Aptamer-antagomiR-NC + ins Aptamer-antagomiR-29b-3p + ins

Arbitrary Units

2

*

Aptamer-antagomiR-NC + ins Aptamer-antagomiR-29b-3p + ins

Arbitrary Units

Arbitrary Units

Aptamer-antagomiR-NC + ins Aptamer-antagomiR-29b-3p + ins 3

120

J

eWAT

+ - + - + - + - - + +

30

*

*

*

Time (min)

Time (min)

H

*

3

*

2

*

*

1 0

p-IR

p-AKT p-GSK3β

Page 33 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 8. Nanocomplex/Aptamer mediated BM-MSCs specific inhibition of miR-29b-3p alleviates the aging-associated insulin resistance (A) The amounts of miR-29b-3p in exosomes released by BM-MSCs of 15-months old

aged

mice

intra–bone

marrow

injected

with

BM-MSCs

specific

nanocomplex/aptamer-antagomiR-29b-3p or nanocomplex/aptamer-antagomiR-NC or vehicle for three months (twice a month); (B) The level of miR-29b-3p in serum derived

exosomes

of

mice

injected

with

aptamer-antagomiR-29b-3p

or

aptamer-antagomiR-NC or vehicle; (C-E) The fasting blood glucose levels, fasting serum insulin, and HOMA-IR index of mice injected with aptamer-antagomiR-29b-3p or aptamer-antagomiR-NC or vehicle; (F-G) The GTTs and ITTs in mice injected with aptamer-antagomiR-29b-3p

or

aptamer-antagomiR-NC

or

vehicle;

(H-J)

Insulin-stimulated IR, AKT, and GSK3β phosphorylation in eWAT, muscle, and liver of mice injected with aptamer-antagomiR-29b-3p or aptamer-antagomiR-NC, respectively (top: representative pictures of western blot, bottom, quantitative measurements of p-IR, p-AKT, and p-GSK3β proteins ). Data are presented as means ± SEM (n= 6-7). Statistical significance was calculated by two-tailed Student's t test or two-way ANOVA, (*P < 0.05).

32


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ASSOCIATED CONTENT Supporting Information: Figure S1. The size and production of exosomes released by BM-MSCs of young mice and aged mice have no significant differences. Figure S2. The basic metabolic index of mice administrated with aged BM-MSC-Exos or young BM-MSC-Exos. Figure S3. The basic metabolic index of mice intra–bone marrow injected with BM-MSCs specific aptamer-agomiR-29b-3p or aptamer-agomiR-NC or vehicle. Figure S4. The delivery efficiency of nanocomplex/aptamer-antagomiR-29b-3p to BM-MSCs of mice is higher by intra-bone marrow injection than intravenous injection. Figure S5. The basic metabolic index of mice intra–bone marrow injected with BM-MSCs specific aptamer-antagomiR-29b-3p or aptamer-antagomiR-NC or vehicle. Figure S6. The function of pancreas is degenerated during ageing, but BM-MSCs specific nanocomplex/Aptamer-antagomiR-29b-3p injection has no significant effects on it.

AUTHOR INFORMATION Corresponding Author: Xiang-Hang

Luo,

Ph.D.&MD,

Professor,

Department

of

Endocrinology,

Endocrinology Research Center, Xiangya Hospital of Central South University, 87# Xiangya Road, Changsha, Hunan 410008, China Tel: +86-0731-89752728 Fax: +86-731-4327324 E-mail: [email protected]

ACKNOWLEDGMENT The authors thank Obio Technology (Shanghai) Corp.,Ltd. for the kindly help in miRNA microarray assays. 33


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Funding: This work was supported by grants from National Natural Science Foundation of China (No.81520108008, 91749105, 81700785, 81801393, 81873643), the Natural Science Foundation of Hunan Province of China (S2019JJQNJJ0681), the Innovation Driven Project of Central South University (grant 20180033040008), and the Talent Plan of Xiangya Hospital at Central South University (grants 52 and 35).

Author contributions: T.S, YZ.X and XH.L designed the experiments and wrote the manuscript; T.S, YZ.X, Y.X, QF.D, JX.W and FL.Z performed most of the experiments; Q.G, CJ.L and Y.H contributed to collect samples. XH.L supervised the experiments and analyzed the data. Competing financial interests: The authors declare no competing financial interests. REFERENCES: (1) Kirkman, M. S.; Briscoe, V. J.; Clark, N.; Florez, H.; Haas, L. B.; Halter, J. B.; Huang, E. S.; Korytkowski, M. T.; Munshi, M. N.; Odegard, P. S.; Pratley, R. E.; Swift, C. S. Diabetes in Older Adults. Diabetes Care. 2012, 35, 2650-2664. (2) Garcia-Garcia, A.; de Castillejo, C. L.; Mendez-Ferrer, S. BMSCs and Hematopoiesis. Immunol Lett. 2015, 168, 129-135. (3) Ugarte, F.; Forsberg, E. C. Haematopoietic Stem Cell Niches: New Insights Inspire New Questions. EMBO J. 2013, 32, 2535-2547. (4) Suchacki, K. J.; Roberts, F.; Lovdel, A.; Farquharson, C.; Morton, N. M.; MacRae, V. E.; Cawthorn, W. P. Skeletal Energy Homeostasis: a Paradigm of Endocrine Discovery. J Endocrinol. 2017, 234, R67-R79. (5) Shimada, T.; Hasegawa, H.; Yamazaki, Y.; Muto, T.; Hino, R.; Takeuchi, Y.; Fujita, T.; Nakahara, K.; Fukumoto, S.; Yamashita, T. FGF-23 Is a Potent Regulator of Vitamin D Metabolism and Phosphate Homeostasis. J Bone Miner Res. 2004, 19, 429-435. (6) Fayed, A.; El, N. M.; Heikal, A. A.; Abdulazim, D. O.; Naguib, M. M.; Sharaf, E. D. U. Fibroblast Growth Factor-23 Is a Strong Predictor of Insulin Resistance among Chronic Kidney Disease Patients. Ren Fail. 2018, 40, 226-230. (7) Lee, N. K.; Sowa, H.; Hinoi, E.; Ferron, M.; Ahn, J. D.; Confavreux, C.; Dacquin, R.; Mee, P. J.; McKee, M. D.; Jung, D. Y.; Zhang, Z.; Kim, J. K.; Mauvais-Jarvis, F.; Ducy, P.; Karsenty, G. Endocrine Regulation of Energy Metabolism by the Skeleton. Cell. 2007, 130, 456-469. (8) Guedes, J. A. C.; Esteves, J. V.; Morais, M. R.; Zorn, T. M.; Furuya, D. T. Osteocalcin Improves Insulin Resistance and Inflammation in Obese Mice: Participation of White Adipose Tissue and Bone. Bone. 2018, 115,68-82. 34


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Bone Marrow Mesenchymal Stem Cells-Derived Exosomal MiR-29b

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