Bone marrow macrophage‐derived exosomal miR‐143‐5p contributes to insulin resistance in hepatocytes by repressing MKP5

Abstract Objective In this study, we aim to explore the role of bone marrow macrophage‐derived exosomes in hepatic insulin resistance, investigate the substance in exosomes that regulates hepatic insulin signalling pathways, reveal the specific molecular mechanisms involved in hepatic insulin resistance and further explore the role of exosomes in type 2 diabetes. Materials and methods High‐fat diet (HFD)‐fed mice were used as obesity‐induced hepatic insulin resistance model, exosomes were isolated from BMMs which were extracted from HFD‐fed mice by ultracentrifugation. Exosomes were analysed the spectral changes of microRNA expression using a microRNA array. The activation of the insulin signalling pathway and the level of glycogenesis were examined in hepatocytes after transfected with miR‐143‐5p mimics. Luciferase assay and western blot were used to assess the target of miR‐143‐5p. Results BMMs from HFD‐fed mice were polarized towards M1, and miR‐143‐5p was significantly upregulated in exosomes of BMMs from HFD‐fed mice. Overexpression of miR‐143‐5p in Hep1‐6 cells led to decreased phosphorylation of AKT and GSK and glycogen synthesis. Dual‐luciferase reporter assay and western blot demonstrated that mitogen‐activated protein kinase phosphatase‐5 (Mkp5, also known as Dusp10) was the target gene of miR‐143‐5p. Moreover, the overexpression of MKP5 could rescue the insulin resistance induced by transfection miR‐143‐5p mimics in Hep1‐6. Conclusion Bone marrow macrophage‐derived exosomal miR‐143‐5p induces insulin resistance in hepatocytes through repressing MKP5.


| INTRODUC TI ON
Insulin resistance is defined as a decrease in the sensitivity and responsiveness of insulin and is the critical pathogenesis of type 2 diabetes. 1 Decreased glycogenesis is a hallmark of insulin resistance in hepatocytes. 2 Overweight and obesity are prevalent in the general population, 3 and obesity has been recognized as a significant risk factor for the development of type 2 diabetes. 4 Compared with normal BMI, the risk of diabetes is increased by 10-40 times for BMI > 30. 5 Obesity causes insulin resistance in a variety of ways, and inflammation caused by obesity is an important factor. 6 Chronic inflammation induced by obesity could lead to hepatic insulin resistance. A significant manifestation of tissue inflammation caused by obesity is the accumulation of macrophages in adipose tissue and the liver. 7 Activated macrophages are usually divided into two categories: M1 macrophages and M2 macrophages. Both M1 macrophages and M2 macrophages are closely related to inflammatory responses, where M1 macrophages are mainly involved in proinflammatory responses and M2 macrophages are mainly involved in anti-inflammatory responses. 8 Studies have found that during obesity, hepatic macrophages towards proinflammatory M1 polarization, which further induces insulin resistance or other liver diseases. 9 Liver macrophages consist of a resident macrophage population, termed as Kupffer cells, and recruited hepatic macrophages, which infiltrate the liver under obese conditions. 10 The accumulation of bone marrow-derived macrophages (BMMs) in adipose tissue is an important cause of insulin resistance in adipose tissue under chronic inflammation resulting from obesity. 11 The evidence also showed that the infiltration of BMMs in the liver was related to liver damage.
The recruitment of BMMs to the liver is regulated by inflammation and fibrosis in the liver. 12,13 Based on this fact, we performed immunofluorescence staining of macrophages in the liver of HFD-fed mice and found that there were more macrophages in the liver of HFD-fed mice compared with chow diet (CD)-fed mice. Based on the previous studies, we thought that the additional macrophages were recruited from the bone marrow. However, the mechanisms by which BMMs from HFD-fed mice participate in the regulation of hepatic insulin resistance remain unknown.
Exosomes are vesicle-like bodies with a diameter of approximately 30-100 nm that are secreted by various types of tissues and cells and play an important role in the pathological and physiological processes in the body. 14 Exosomes can transmit information in cells by carrying various substances, such as proteins, RNA and lipids. 15 MicroRNAs are a class of noncoding small RNA molecules with a length of 22 nucleotides. MicroRNAs play an important role in the posttranscriptional regulation of metabolic diseases. 16 In recent years, many studies have found that microRNAs carried by exosomes play an important role in insulin resistance. An article published in Cell demonstrated that exosomes derived from adipose tissue macrophages could regulate the insulin sensitivity of liver and muscle cells. Further research by this team showed that the miR-155 carried by exosomes impaired insulin sensitivity. 17 Similarly, studies also found that adipose tissue-derived exosomes carrying miR-27a induce insulin resistance in skeletal muscle by inhibiting PPARγ. 18 These studies indicated that microRNAs carried by exosomes played an important role in insulin resistance.
Here, we found that BMM-derived exosomes from HFD-fed mice were able to induce insulin resistance in the mouse hepatic cell line Hep1-6. Mechanistically, miR-143-5p was significantly increased in the BMM-derived exosomes of HFD-fed mice, and MKP5 functions as the downstream target of exosomal miR-143-5p in the regulation of insulin resistance. Taken together, our study shows the role of BMM-derived exosomes from HFD-fed mice in regulating obesity-associated insulin resistance. Moreover, miR-143-5p in exosomes released by BMMs may be a promising target for treating obesity-related insulin resistance. 8-week-old male wild-type (WT) C57BL/6J mice were purchased from SPF Biotechnology Co. Ltd. The mice were separated into two groups (eight mice in each group) and fed a standard chow diet or a high-fat diet (HFD, 45% kcal from fat from Medicine) for 12 weeks. All mouse procedures were approved by the Ethics and Animal Welfare Committee at College of Life Sciences, Beijing Normal University.

| Histological analysis of tissues
The liver specimens were embedded in O.C.T. (Tissue-Tek) and cut into a thickness of 6 µm. For Oil Red O staining, the slides were incubated with Oil Red O (Solarbio) at 37℃ for 30 min, then used 60% 1,2-propanediol to wash. 19 For haematoxylin and eosin (H&E) staining, first, the slides were stained with haematoxylin for 5 min, then washed with 1% ethanol hydrochloride for 3 s. After water washing, the slides were incubated with eosin for 3 min and dehydrated with an alcohol gradient. 20

| Cell culture
The mouse hepatic cell line Hep1-6 and macrophage cell line RAW 264.7 were purchased from the American Type Culture Collection.

| Isolation and cultivation of mice BMMs
Cells were obtained from the tibia and femur bone marrow of chow diet-fed and HFD-fed mice and were cultured in the presence of L-929 conditioned medium as described in detail previously. 12 The identification of BMMs is assessed using immunocytochemistry analysis of F4/80 expression.

| Oleic acid/palmitic acid (O/P) treatment
The method was performed as previously described. 21 Oleic acid of

| Exosome purification and characterization
After culturing for 72 h, debris and dead cells in the BMM medium were removed by centrifugation at 2000 × g for 20 min and then filtrated through the 0.2 μm filter. The medium was then subjected to ultracentrifugation at 100,000 × g for 4 h at 4°C. After washing with PBS (100,000 × g for 20 min), the exosome-containing pellet was resuspended in PBS. 17 The characterization of exosomes was confirmed by measuring the expression of exosome-specific markers FLOT1, CD63 and CD9 by western blot analysis.

| Exosomes tracing
To monitor the exosome trafficking, exosomes were labelled with PKH26 fluorescent dye using the PKH26 fluorescent cell linker kit (Sigma-Aldrich). After PKH26 staining, the exosomes were washed in PBS and collected by ultracentrifugation (100,000 × g for 20 min) at 4°C. Finally, PKH26 labelled exosomes were resuspended in PBS. 17

| Electron microscopy
Exosomes obtained by ultracentrifugation were sent to the Central Laboratory of Peking Union Medical College for electron microscope examination.

| RNA extraction and real-time PCR
The total RNA was extracted from liver tissues and Hep1-6 cells using TRIzol reagent (Invitrogen). Real-time PCR was performed using IQ5 system (Bio-Rad). The primers used for reverse transcription and real-time PCR were listed in Table 1 and Table 2.

| Cell transfection
The mimics and inhibitors of miR-143-5p, MKP5 siRNA and negative control siRNA (NC) were purchased from GenePharma. Mimics, inhibitors, siRNA or miR-negative control were transfected for 48 h using HiPerFect transfection reagent (Qiagen) according to the manufacturer's protocol.

| Protein extraction and western blot analysis
RIPA buffer (Solarbio) was used for protein extraction of liver tissues and Hep1-6 cells. The blot was incubated with HRP-conjugated anti-IgG, followed by the detection with ECL (Millipore). Antibodies

| Glycogen content measurement
Glycogen content in cells or livers was analysed using a glycogen assay kit (Biovision), according to the manufacturer's instructions.

| Co-culture assay
After the treatment with O/P, RAW264.7 cells were washed with PBS twice and co-cultured with Hep1-6 cells at a ratio of 1:1 using

| Immunofluorescence
The slides were fixed to the samples in 4% paraformaldehyde for 30 min at room temperature. Then, the slides were stained with immunofluorescence as previously described. 22

| Statistical analysis
The data represent the mean ± standard error of the mean (SEM).
The two-tailed unpaired student's t-test was used to compare the differences of the two groups. The ANOVA multiple comparisons test followed by the Turkey post hoc test were used for comparisons of two more groups. p < 0.05 was considered to indicate a statistically significant difference.

| HFD-fed mice developed obesity and insulin resistance accompanied by increased macrophage infiltration in the liver
Compared with chow diet (CD)-fed mice, the weight of HFD-fed mice was significantly higher ( Figure 1A). Oil Red O and H&E staining revealed a significant increase in hepatic lipid deposition in the livers of the HFD-fed mice ( Figure 1B). Notably, the phosphorylation of AKT and GSK was reduced in the livers of HFD-fed mice, indicating impaired activation of the AKT/GSK pathway ( Figure 1C).
Moreover, the high-fat diet also reduced glycogen synthesis in the livers of mice ( Figure 1D). These data indicated that HFD-fed mice developed obesity and insulin resistance. Additionally, the results of the immunofluorescence assay and western blot showed that macrophage infiltration was significantly increased in the livers of HFDfed mice ( Figure 1E,F). BMMs from HFD-fed mice were polarized TA B L E 2 The real-time PCR primer sequence Gene name Forward primer Reverse primer  Figure 1G) and low expression of Cd206, Il-10 and Arginase-1( Figure 1H). Further, we also detected the polarization of macrophages in the liver of mice.
Our results showed that compared with CD-fed mice, macrophages in the liver of HFD-fed mice were polarized to M1 with the high expression of Tnfα, Il-6 and Mcp-1, but no change of M2 marker Cd206, and Il-10 and low expression of Arginase-1 ( Figure 1I).

cells induced insulin resistance in Hep1-6 cells
To verify the relationship between the BMMs in HFD-fed mice and liver cells, we obtained BMMs from HFD-fed mice and CD-fed mice.
We collected the cell supernatant and centrifuged it to remove the cell debris. As shown in Figure 2A, were co-cultured using a transwell (membrane pore = 0.4 μm) plate.
The activation of AKT and GSK were reduced in Hep1-6 cells, accompanied by decreased glycogenesis (Figure 2E,F). These data indicated that the BMMs from HFD-fed mice and O/P-treated RAW264.7 cells secreted some factor that could induce hepatic insulin resistance.

| Exosomes secreted by BMMs of HFD-fed mice and O/P-treated RAW264.7 cells mediated hepatocyte insulin resistance
Two types of cells that can interact via exosomes have been identified. Therefore, we speculated that exosomes secreted by the

| Exosomes secreted by the BMMs of HFD-fed mice induced insulin resistance in Hep1-6 cells by delivering miR-143-5p
It has been reported that exosomes could affect the biological function of recipient cells by delivering microRNAs. Therefore, a micro-RNA array was performed to detect the levels of microRNAs in the exosomes secreted by the BMMs of HFD-fed mice and CD-fed mice.
The results showed 32 increased microRNAs in the exosomes secreted by the BMMs of HFD-fed mice compared with the BMMs of CD-fed mice ( Figure 4A). To confirm the array results, the changes in upregulated microRNAs were examined using a real-time PCR. As shown in Figure 4B,C, miR-143-5p was significantly upregulated in the BMMs of HFD-fed mice and their exosomes. The level of miR-143-5p was much higher in macrophages compared with hepatocytes (Approximately 15 times)( Figure S1). Moreover, we found

| Mkp5 was identified as a target of miR-143-5p
We next identified the potential target of miR-143-5p involved in glycogen synthesis using computational miRNA target prediction databases. The results from TargetScan revealed that there is a binding site of miR-143-5p on the 3′UTR of Mkp5 ( Figure 5A). To further verify that Mkp5 is the target of miR-143-5p, we cloned the 3′UTR of Mkp5 containing the predicted binding site and its mutation site ( Figure 5B) into the pmirGLO vector. As shown in Figure 5C

| Mkp5 participated in hepatic insulin resistance
In this study, we found that the protein level of MKP5 was significantly decreased in the livers of HFD-fed mice ( Figure 6A). In addition, the immunofluorescence assay showed that the MKP5 was  Figure 6E). Taken together, these findings indicated that miR-143-5p-mediated insulin resistance by suppressing MKP5 expression.

| DISCUSS ION
In this study, we identified that in the liver of HFD-fed mice, BMMs were polarized towards the M1 state, which induced hepatic insulin resistance by secreting exosomes.
Studies have found that macrophages play an important role in liver inflammatory fibrosis and chronic injury. In the injured liver, macrophages recruited from bone marrow could regulate liver inflammation and fibrosis. 23  Obesity could induce adipose tissue macrophages to differentiate into the proinflammatory M1 type, which is related to the inflammation produced by adipose tissue and insulin resistance. 26 Our results showed that in the livers of HFD-fed mice, the activation of the insulin signalling pathway was impaired, glycogen synthesis was reduced, and infiltration of macrophages was significantly increased.
The increased macrophages in the liver were recruited from bone marrow cells. Furthermore, compared with the BMMs of CD-fed mice, the BMMs of HFD-fed mice were obviously polarized towards M1 proinflammatory macrophages. This polarization further led to the appearance of inflammation in the body.
To determine the relationship between M1 BMMs and hepatocytes, we speculated that exosomes secreted by these BMMs might

CO N FLI C T O F I NTE R E S T
The authors declare no conflict or financial interest.